Transcriptomic Changes in Adult Hypothalamic Neurons Following Leptin Sensitivity Modulation

Modulating leptin sensitivity in hypothalamic neurons is critical for metabolic regulation and plays a key role in the development of obesity. This study examines the effects of three distinct approaches—leptin exposure (LEPA), leptin antagonist administration (LANTA), and palmitate treatment (PA)—on adult-derived mHypoA-2/12 neurons. Transcriptomic analysis using 3' mRNA-Seq revealed significant downregulation of several pathways, including the NOD-like receptor signaling pathway, C-type lectin receptor signaling pathway, NF-κB signaling pathway, and IL-17 signaling pathway. These pathways are closely linked to neuroinflammatory processes, suggesting that modulating leptin sensitivity may influence inflammation in the hypothalamus. The observed downregulation indicates a complex interaction between leptin signaling and cellular stress responses, which may represent an adaptive mechanism to prolonged exposure to leptin or fatty acids. Understanding these dynamics is crucial for gaining insight into how neuroinflammation contributes to the development of leptin resistance and related metabolic disorders. The adult mouse hypothalamic cell line mHypoA-2/12 (Clu177, Cedarlane, Canada) was cultured in Dulbecco's Modified Eagle's Medium with GlutaMAX (DMEM, 4500 mg/L, Gibco, Thermo Fisher Scientific, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% Antibiotic-Antimycotic solution (containing 10,000 units/mL penicillin, 10,000 µg/mL streptomycin, and 25 µg/mL amphotericin B, Gibco). Cells were seeded at a density of 1.5x10⁵ cells per well in 24-well plates and incubated at 37°C with 5% CO₂. For treatments, cells were exposed to 200 nM leptin (LEPA, PLR, Israel), 30 µM leptin antagonist (LANTA, PLR, Israel), or 5 mM palmitic acid (PA, Merck, USA), which was conjugated with fatty acid-free bovine serum albumin (NEFA-free BSA) and dissolved in serum-free DMEM, based on the method of Mayer and Belsham (2010). Vehicle controls were prepared with serum-free DMEM and 5% NEFA-free BSA (CTR). After treatment, cells were harvested for analysis using the RealTime-Glo™ MT Cell Viability Assay for viability, the Caspase-Glo® 3/7 Assay for apoptosis, and the Fluo-4 NW Calcium Assay Kit for calcium measurement.