Fig. 1: Transcriptomic profiles of AgRP neurons during opposing states of energy balance.

a Schematic of the experimental design. NuTRAP^AgRP mice were fed, then a subset were fasted overnight (~19 hr), following which mice were treated with IP vehicle or leptin (5 mg/kg). After 3 hours, mice were euthanized and ARCs were isolated, frozen, and homogenized according to the TRAP protocol. b Representative immunofluorescence image of ARC revealing induction of pSTAT3 45-minutes following leptin administration (right) compared to vehicle treatment (left); repeated independently 2 times. c Scatter plot showing comparing expression of genes within AgRP neurons from fasted versus fed littermates (n = 4, 3) [fold change >0.5 up (red) or down (blue), false discovery rate (FDR) < 0.05]; CPM, counts per million. The number of genes in a given expression profile is in parentheses. d Heatmap of differentially expressed genes in fed, fasted, and leptin-treated mice. e WebGestalt (WEB-based Gene SeT AnaLysis Toolkit) gene set enrichment analysis (GSEA) for geneontology biological process in either fasting-regulated (left, induced and repressed), or leptin-regulated (right, induced and repressed) transcriptional pattern categories with Normalized Enrichment Scores. All bars have an FDR < 0.05 except those that are light blue in the leptin-regulated condition f Scatter plot showing regulated genes within AgRP neurons from leptin-treated versus fasted littermates (n = 4, 4) (fold change > 0.5 up (red) or down (blue), FDR < 0.05). CPM, counts per million. FDR-adjusted p-values with a threshold of 0.05 were generated using the Benjamini-Hochberg method to account for multiple comparisons. The number of genes in a given expression profile is in parentheses. g Heatmaps of selected gene categories. h Top: bar graphs showing the number of fasted up-regulated genes (N = 527) that are also down-regulated with leptin (blue bars, N = 90). Bottom: bar graphs showing the number of fasted down-regulated genes (N = 295) that are also up-regulated with leptin (blue bars, N = 30). i Genome browser views (in Integrative Genomics Viewer (IGV)) of RNA-seq tracks of representative fasting-upregulated, fasting-downregulated, leptin-upregulated, leptin-downregulated, bidirectional fasting upregulated (Bi-fasting upregulated), and bidirectional leptin upregulated (Bi-leptin upregulated) genes. The data from TRAP-seq experiments was generated from three independent biological replicates for fed mice, four independent biological replicates for fasted mice, and four independent biological replicates for leptin-treated mice.