Fig. 3. Celastrol activates hypothalamic leptin signaling in diet-induced obese (DIO) rats without affecting serum leptin levels. (A) Principle component (PC) analysis of RNA-seq data from the hypothalami of rats after 3 weeks of treatment (n=3 for each group). (B) The hierarchical clustering of differentially expressed genes (left panel) and enriched pathways, as identified by ingenuity pathway analysis (right panel). (C) Western blot of the protein levels of downstream effectors of leptin, namely, phosphorylated-signal transducer and activator of transcription 3 (p-STAT3)^Tyr705 and total STAT3, and β-actin, in the hypothalami of vehicle-, antibiotic-, celastrol-, and celastrol+antibiotic-treated rats. (D) The ratio of the signal intensity of p-STAT3 to that of total STAT3. (E) The serum leptin levels, as determined by enzyme-linked immunosorbent assay (ELISA), after celastrol treatment for 1, 2, and 3 weeks. (F) The mRNA expression of critical genes in the leptin signaling pathway, as determined by quantitative real-time polymerase chain reaction (RT-qPCR). The findings indicate that the activation of the leptin signaling pathway might result from an improvement in leptin sensitivity. The error bars represent the standard error of means. (E, F) The P values were determined by Student's t-test. DARPP32 (PPP1R1B), protein phosphatase 1 regulatory inhibitor subunit 1B; cAMP, cyclic adenosine monophosphate; GNRH, gonadotropin releasing hormone; CXCR4, C-X-C motif chemokine receptor 4; GABA, gamma-aminobutyric acid; nNOS, nitric oxide synthase; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; ERK, extracellular signal-regulated kinases; MAPK, mitogen-activated protein kinases; ErbB (EGFR), epidermal growth factor receptor; PXR (NR1I2), nuclear receptor subfamily 1 group I member 2; IGF-1, insulin like growth factor 1; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; VDR, vitamin D receptor; NS, not significant; Agrp, agouti-related peptide; Npy, neuropeptide Y; Pomc, pro-opiomelanocortin; Ptpn1, protein tyrosine phosphatase non-receptor type 1; Socs3, suppressor of cytokine signaling 3.