ABSTRACT: Background: Leptin is a master regulator of energy homeostasis, acting through its long‑form receptor LepRb to modulate neuronal activity, transcriptional programs, and metabolic pathways that control feeding and body‑weight regulation. While classical work established JAK2–STAT3 signaling as a central axis of leptin action, it is now clear that leptin triggers a temporally layered response, in which early rapid signaling events (e.g., STAT3 phosphorylation) are followed by late transcriptional and pathway‑level remodeling that can reshape neuronal plasticity, synaptic architecture, and long‑term metabolic outcomes. However, dissecting these temporal dynamics in vivo is extremely challenging because LepRb‑expressing neurons are heterogeneous, sparse, and distributed across multiple brain regions. As a result, resolving early vs. late transcription factor activity, pathway‑level dynamics, and interaction effects between regulators requires a controlled model system with sufficient statistical power. This project directly addresses this gap by using a hypothalamic cell line engineered to overexpress LepRb, enabling precise stimulation with leptin and systematic high‑resolution profiling of transcriptional and proteomic responses. By comparing wild‑type cells with CRISPR‑mediated Stat3 or Etv6 deletions, the project uncovers how transcriptional regulators contribute to the architecture of leptin signaling across time. Proteomics is applied to compare, and validate, transcriptome findings.Experimental Workflow: Cells: Clu177 hypothalamic cells were cultured in high‑glucose DMEM supplemented with 10% heat‑inactivated FBS and 1% Pen/Strep. Cells were transfected with the pCAG‑2A‑H2B‑Venus_mOBRb‑HA plasmid using FuGene® HD, and were selected 24 h later with 3 µg/ml puromycin. High‑LepRb–expressing Clu177mOBRb cells were isolated by FACS based on GFP signal and were maintained in full medium plus puromycin. Leptin Exposure: For stimulation assays, cells were starved for 2 h in DMEM containing 0.1% FBS. Recombinant murine leptin was added at concentrations ranging from 10 pM to 100 nM, and cells were stimulated for defined early and late time points. After stimulation, cells were washed with ice‑cold PBS and were either snap‑frozen at −80 °C for RNA/protein preparation. Crispr-Cas9-mediated Stat3 and Etv6 deletion: Deleted Clu177mOBRb cell lines were generated via a CRISPR–Cas9 dual‑guide strategy. sgRNAs were designed using CRISPOR (https://crispor.gi.ucsc.edu/), and cells were electroporated with pX330 constructs encoding Cas9 and paired sgRNAs. Edited cells were selected with 10 µg/ml blasticidin for 48 h; single clones were expanded, and genomic deletions were confirmed by PCR.
Paul Pfluger