Leptin Receptor Signaling Regulates Protein Synthesis Pathways and Neuronal Differentiation in Pluripotent Stem Cells

The role of leptin receptor (OB-R) in linking pluripotency with growth, development, and the consequences on the progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying db/db mutation exhibited metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) demonstrated alterations in the expression of proteins involved in embryonic development. An upregulation in the expression of eukaryotic translation initiation factor4e (eIF4e) and Stat3 binding to the eIF4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs towards the neuronal lineage showed defects compared to controls. Gene editing to correct point mutation in db/db iPSCs using CRISPR/Cas9, restored the metabolic defects, expression of neuronal markers, and protein synthesis as corroborated by RNAseq analyses. These data imply a direct role for OB-R in regulating metabolic properties in embryonic fibroblasts and key developmental pathways in iPSCs. Overall design: Embryonic day 14.5 wild type control (Ctrl) (N=4) and db/db MEFs (N=4) were derived from breeding OB-Rdb/+ heterozygous mice (Jackson Laboratory Inc.). Generation of mouse iPSCs involved infection of primary MEFs with mouse STEMCCA lentivirus vector expressing the reprogramming factors Oct4, Sox2, Klf4 and cMyc. Gene-correction in db/db iPSCs was performed using CRISPR-Cas9. Total RNA was isolated from iPSCs, Ctrl (N=4), db/db (N=4), and db/Corr (N=2) in duplicates by using RNeasy kit (Qiagen) and RNA sequencing was performed by using Illumina Novaseq6000.