BVT.2733, a Selective 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor, Attenuates Obesity and Inflammation in Diet-Induced Obese Mice

BACKGROUND: Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro. CONCLUSIONS/SIGNIFICANCE: These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease

Nuclear receptor modulators inhibit osteosarcoma cell proliferation and tumour growth by regulating the mTOR signaling pathway

Abstract Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Chemoresistance leads to poor responses to conventional therapy in patients with osteosarcoma. The discovery of novel effective therapeutic targets and drugs is still the main focus of osteosarcoma research. Nuclear receptors (NRs) have shown substantial promise as novel therapeutic targets for various cancers. In the present study, we performed a drug screen using 29 chemicals that specifically target 17 NRs in several different human osteosarcoma and osteoblast cell lines. The retinoic acid receptor beta (RARb) antagonist LE135, peroxisome proliferator activated receptor gamma (PPARg) antagonist T0070907, liver X receptor (LXR) agonist T0901317 and Rev-Erba agonist SR9011 significantly inhibited the proliferation of malignant osteosarcoma cells (U2OS, HOS-MNNG and Saos-2 cells) but did not inhibit the growth of normal osteoblasts. The effects of these NR modulators on osteosarcoma cells occurred in a dose-dependent manner and were not observed in NR-knockout osteosarcoma cells. These NR modulators also significantly inhibited osteosarcoma growth in vivo and enhanced the antitumour effect of doxorubicin (DOX). Transcriptomic and immunoblotting results showed that these NR modulators may inhibit the growth of osteosarcoma cells by regulating the PI3K/AKT/mTOR and ERK/mTOR pathways. DDIT4, which blocks mTOR activation, was identified as one of the common downstream target genes of these NRs. DDIT4 knockout significantly attenuated the inhibitory effects of these NR modulators on osteosarcoma cell growth. Together, our results revealed that modulators of RARb, PPARg, LXRs and Rev-Erba inhibit osteosarcoma growth both in vitro and in vivo through the mTOR signaling pathway, suggesting that treatment with these NR modulators is a novel potential therapeutic strategy

Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux.

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation

Effect of 11β-HSD1 on the inflammation gene expression in PA or LPS-activated J774A.1 macrophages <i>in vitro</i>.

<p>A, J774.1 macrophages were treated with palmitic acid (PA) (50−200 µmol/L) or LPS (100 ng/ml) for 24 h. B–C, J774.1 macrophages were activated by PA (200 µmol/L) or LPS (100 ng/ml) and co-treated with 11β-HSD1 inhibitor BVT.2733 (25−100 µmol/L) for 24 h. D–G, J774.1 macrophages were transfected with either sh-RNA for mouse 11β-HSD1 (sh-HSD1) or a negative control (sh-con) by Lentivirus. After 72 h incubation, cells were treated with PA (200 µmol/L) or LPS (100 ng/ml) for 24 h. Efficiency of 11β-HSD1 knockdown on mRNA level (D) and protein level (E). Effects of knockdown of 11β-HSD1 on MCP-1, IL-6, and TNF-α expression in PA (F) or LPS (G) treated macrophages. H–K, J774.1 macrophages were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. After 72 h incubation, cells were treated with PA (100 µmol/L) or LPS (50 ng/ml) and co-treated with 11β-HSD1 inhibitor BVT.2733 for 24 h. Efficiency of 11β-HSD1 overexpression on mRNA level (H) and protein level (I). Effects of overexpression of 11β-HSD1 on MCP-1, IL-6, and TNF-α expression in PA (J) or LPS (K) treated macrophages. mRNA for IL-6, MCP-1 and TNF-α were determined by real-time PCR, protein of 11β-HSD1 were determined by Western blot. The results are shown as the means ± SEM of three individual experiments. *<i>P</i><0.05; **<i>P</i><0.01 vs con (B, C, F, G) or sh-con (D) or emp (H) or emp+PA (J) or emp+LPS (K). # <i>P</i><0.05; ## <i>P</i><0.01 vs PA (B) or LPS (C) or sh-con+PA (F) or sh-con+LPS (G) or HSD1+PA (J) or HSD1+LPS (K).</p

Effect of 11β-HSD1 on the inflammation gene expression in PA or LPS-activated 3T3-L1 preadipocytes <i>in vitro</i>.

<p>3T3-L1 preadipocytes were activated by PA (200 µmol/L) (A) or LPS (200 ng/ml) (D) and co-treated with 11β-HSD1 inhibitor BVT.2733 (50−100 µmol/L) for 24 h. 3T3-L1 preadipocytes were transfected with either sh-RNA for mouse 11β-HSD1 (sh-HSD1) or a negative control (sh-con) by Lentivirus. Cells were treated with PA (200 µmol/L) (B) or LPS (200 ng/ml) (E) for 24 h. 3T3-L1 preadipocytes were transfected with the expression vector for 11β-HSD1 (HSD1) or a corresponding empty vector (emp) using Lentivirus. Cells were treated with PA (200 µmol/L) (C) or LPS (200 ng/ml) (F) for 24 h. mRNA for IL-6, MCP-1 and TNF-α were determined by real-time PCR. The results are shown as the means ± SEM of three individual experiments. *<i>P</i><0.05; **<i>P</i><0.01 vs con (A, B, D, E) or or emp+PA (C) or emp+LPS (F). # <i>P</i><0.05 vs PA (A) or LPS (D) or sh-con+PA (B) or sh-con+LPS (E) or HSD1+PA (C) or HSD1+LPS (F).</p

Effect of HFD and BVT.2733 treatment on the number of macrophages in SVF of epididymal fat tissue.

<p>Cells in the SVF of epididymal fat tissue from three groups of mice were analyzed using flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040056#s4" target="_blank">METHODS</a> section. A, Representative flow cytometric profiles of cells in the SVF of epdidymal fat tissue derived from NC, HFD, and HFD+BVT mice individually. B, The cell number in F4/80-positive fraction. C, F4/80-positive/CD11b-positive/CD11c- negetive fraction. D, F4/80-positive/CD11b-positive/CD11c- positive fraction. The results are shown as the means ± SEM. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with NC group; #, <i>P</i><0.05; ##, <i>P</i><0.01 compared with HFD group. n = 3−4 animals per group.</p