Immortalization and Characterization of GFAP-expressing Glial Cells from the Adult Mouse Hypothalamus, Cortex, and Brain Stem.

The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1, Aqp4, Glt1 and S100b, and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b, Tnfα and Cxcl5 in mHypoA-Ast1 cells after 4 h, while 50 µM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research.

Bisphenol A induces miR-708-5p through an ER stress-mediated mechanism altering neuronatin and neuropeptide Y expression in hypothalamic neuronal models.

Bisphenol A (BPA) is an endocrine disrupting chemical that promotes obesity. It acts on the hypothalamus by increasing expression of the orexigenic neuropeptides, Npy and Agrp. Exactly how BPA dysregulates energy homeostasis is not completely clear. Since microRNAs (miRNA) have emerged as crucial weight regulators, the question of whether BPA could alter hypothalamic miRNA profiles was examined. Treatment of the mHypoA-59 cell line with 100 µM BPA altered a specific subset of miRNAs, and the most upregulated was miR-708-5p. BPA was found to increase the levels of miR-708-5p, and its parent gene Odz4, through the ER stress-related protein Chop. Overexpression of an miR-708-5p mimic resulted in a reduction of neuronatin, a proteolipid whose loss of expression is associated with obesity, and an increase in orexigenic Npy expression, thus potentially increasing feeding through converging regulatory pathways. Therefore, hypothalamic exposure to BPA can increase miR-708-5p that controls neuropeptides directly linked to obesity.

Mechanisms Driving Palmitate-Mediated Neuronal Dysregulation in the Hypothalamus.

The hypothalamus maintains whole-body homeostasis by integrating information from circulating hormones, nutrients and signaling molecules. Distinct neuronal subpopulations that express and secrete unique neuropeptides execute the individual functions of the hypothalamus, including, but not limited to, the regulation of energy homeostasis, reproduction and circadian rhythms. Alterations at the hypothalamic level can lead to a myriad of diseases, such as type 2 diabetes mellitus, obesity, and infertility. The excessive consumption of saturated fatty acids can induce neuroinflammation, endoplasmic reticulum stress, and resistance to peripheral signals, ultimately leading to hyperphagia, obesity, impaired reproductive function and disturbed circadian rhythms. This review focuses on the how the changes in the underlying molecular mechanisms caused by palmitate exposure, the most commonly consumed saturated fatty acid, and the potential involvement of microRNAs, a class of non-coding RNA molecules that regulate gene expression post-transcriptionally, can result in detrimental alterations in protein expression and content. Studying the involvement of microRNAs in hypothalamic function holds immense potential, as these molecular markers are quickly proving to be valuable tools in the diagnosis and treatment of metabolic disease.