Supplementary Fig. 3
mRNA and protein levels in hypothalamus. (A) Heatmap clustering of hypothalamus samples from vehicle and celastrol treated rats based on RNA-seq analysis. (B) Normalized counts of genes involved in leptin signaling pathway. (C) Western blot analysis and (E) intensity analysis of phosphorylated protein kinase-like endoplasmic reticulum kinase (PERK) and total PERK protein in hypothalamus from vehicle, vehicle+antibiotics, celastrol, celastrol+antibiotics treated rats. (D) mRNA expression level of X-box binding protein 1 (Xbp1). (F) mRNA expression level of Perk. Error bars are represented as mean±standard error of mean. (D, F) P values were determined by Student's t-test. Adcy5, adenylate cyclase 5; Agrp, agouti-related peptide; Foxo1, forkhead box O1; Jak2, Janus kinase 2; Lepr, leptin receptor; Npy, neuropeptide Y; Pik3r1, phosphoinositide-3-kinase regulatory subunit 1; Plcb1, phospholipase C beta 1; Pomc, pro-opiomelanocortin; Prkacb, protein kinase cAMP-activated catalytic subunit beta; Socs3, suppressor of cytokine signaling 3; Stat3, signal transducer and activator of transcription 3; NS, not significant.
dmj-2019-0124-s004.pdf
Supplementary Fig. 4
Food intake of different genotypes of diet-induced obese (DIO) rats. Daily food intake of wild-type (WT) DIO rats in (A) 1st week, (B) 2nd week, and (C) 3rd week after treatment of celastrol. Daily food intake of heterozygous leptin knockout (Lep−/+) DIO rats in (D) 1st week, (E) 2nd week, and (F) 3rd week after treatment of celastrol. Daily food intake of homozygous leptin knockout (Lep−/−) DIO rats in (G) 1st week, (H) 2nd week, and (I) 3rd week after treatment of celastrol. Error bars are represented as mean±standard error of mean. P values were determined by one-way analysis of variance (ANOVA) or Student's t-test. NS, not significant. aP<0.05.
dmj-2019-0124-s005.pdf
Supplementary Fig. 7
Blood biochemical test after toxicity test with celastrol in mice. (A) Alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) total protein (TP), (D) albumin (ALB), (E) total bilirubin (TBIL), (F) alkaline phosphatase (ALP), (G) glucose (GLU), (H) blood urea nitrogen (BUN), (I) creatine (CREA), (J) uric acid (UA), (K) calcium (Ca), (L) phosphorus (P), (M) cholesterol (CHOI), (N) triglycerides (TG), (O) high density lipoprotein cholesterol (HDL-C), (P) low density lipoprotein cholesterol (LDL-C), (Q) lactate dehydrogenase (LDH), (R) creatine kinase (CK), (S) creatine kinase isoenzyme (CKMB), and (T) superoxide dismutase (SOD). Error bars are represented as mean±standard error of mean. P values were determined by analysis of variance (ANOVA) test. NS, not significant.
dmj-2019-0124-s008.pdf
Fig. 1Celastrol protects rats against diet-induced obesity. Diet-induced obese (DIO) Sprague-Dawley rats were orally administered vehicle or celastrol (500 µg/kg) every day for 3 weeks. (A) The body weight and (B) percent decrease (%) in the body weight of the DIO rats during the treatment period (n=8 for vehicle group; n=7 for celastrol group). (C) Representative H&E staining of abdominal white adipose tissue (WAT) and the liver. The results of the oral glucose tolerance test (OGTT) of the rats before (D) and after (E) the treatment period. (F) The serum triglyceride levels of the rats after 3 weeks of treatment. The results of the insulin tolerance test (ITT) of the rats before (G) and after (H) the treatment period. (I) The serum cholesterol levels of the rats after 3 weeks of treatment. The error bars represent the standard error of means. The P values were determined by (B) Dunnett's post hoc test or (D–I) Student's t-test. NS, not significant. aP<0.05.
Fig. 2Celastrol alters the microbiota composition in diet-induced obese (DIO) rats. The microbiota composition of the feces of vehicle-, celastrol-, and celastrol+antibiotic-treated DIO rats was analyzed by 16S rRNA sequencing (n=6 for each group). (A) The weighted version of the UniFrac-based principal coordinate analysis (PCoA), (B) α-diversity, and (C) relative abundance (%) of the microbiota at the phylum level in vehicle- and celastrol-treated rats after 3 weeks of treatment. (D, E) The consecutive relative abundance of the microbiota of (D) all phyla, (E) Bacteroidetes (left panel), and Firmicutes (right panel) in vehicle- and celastrol-treated rats after 1, 2, and 3 weeks of treatment. (F) To understand the role of the microbiota in the anti-obesity effect of celastrol, antibiotics were added to the drinking water beginning the second week of celastrol treatment, as indicated by antibiotic intervention in the schematic diagram (n=7 for the celastrol group; n=8 for the antibiotic intervention group). (G) The α-diversity and (H) weighted version of the UniFrac-based PCoA of the microbiota in celastrol- and celastrol+antibiotic-treated rats after 3 weeks of treatment. (B, G) The P values were determined by the rank sum test. Alterations in the relative abundances of Bacteroidetes and Firmicutes were fitted by linear regression.
Fig. 3Celastrol 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.
Fig. 4A higher dose of celastrol protects Lep−/+ rats, but not Lep−/− rats, against high-fat-diet-induced obesity. Heterozygous (Lep−/+) and homozygous (Lep−/−) leptin knockout diet-induced obese rats were orally administered vehicle, 500 µg/kg celastrol or 1,000 µg/kg celastrol every day for 3 weeks. (A) The body weight and (B) the percent decrease (%) in body weight of the Lep−/+ rats during the treatment period (n=9 for the vehicle group; n=7 for the 500 µg/kg celastrol group; n=8 for the 1,000 µg/kg celastrol group). (C) The body weight and (D) the percent decrease (%) in body weight of the Lep−/− rats during the treatment period (n=5 for the vehicle group; n=6 for the 1,000 µg/kg celastrol group). The error bars represent the standard error of means. The P values were determined by one-way analysis of variance (ANOVA) or Student's t-test. aP<0.05.
Fig. 5Celastrol regulates energy homeostasis by enhancing energy expenditure (EE). (A–C) The average daily food intake of the (A) wild-type (WT), (B) Lep−/+, and (C) Lep−/− diet-induced obese (DIO) rats during the 3 weeks of treatment. (D-K) The WT DIO rats were placed in TSE Labmaster Caging System metabolic cages and administered vehicle or 500 µg/kg celastrol for 3 days after 1 week of celastrol acclimation (n=3 for each group). The dynamic or cycle-summarized (D, G) respiratory exchange ratios (RERs), (E, H) EE, and (F, I) the level of physical activity of each group of rats. (J, K) The mRNA expression of genes that participate in adipogenesis and fatty acid metabolism pathways in (J) white adipose tissue (WAT) and (K) brown adipose tissue (BAT). The error bars represent the standard error of means. (A-C and G-K) The P values were determined by Student's t-test. NS, not significant. aP<0.05, bP<0.01.
Fig. 6Celastrol enhances energy expenditure (EE) via the leptin signaling pathway. To prevent the effects of different body weights on EE, wild-type (WT) and Lep−/− rats were fed a strictly controlled high-fat diet (HFD) to maintain equal body weights between the two groups. (A) A schematic diagram of diet control and (B) the body weight of the WT and Lep−/− rats before treatment with celastrol. (C–H) The WT and Lep−/− rats were placed in TSE Labmaster Caging System metabolic cages and administered vehicle or 500 µg/kg celastrol for 3 days after 1 week of celastrol acclimation (n=5 for each group). (C, F) The dynamic or cycle-summarized respiratory exchange ratios (RERs), (D, G) EE, and (E, H) the level of physical activity of each group of rats. The error bars represent the standard error of means. (B and F–H) The P values were determined by Student's t-test. NS, not significant. aP<0.05, bP<0.0001.