International Journal of Obesity (2014) 38, 1432–1439 © 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo ORIGINAL ARTICLE 4β-Hydroxywithanolide E isolated from Physalis pruinosa calyx decreases inflammatory responses by inhibiting the NF-κB signaling in diabetic mouse adipose tissue T Takimoto, Y Kanbayashi, T Toyoda, Y Adachi, C Furuta, K Suzuki, T Miwa and M Bannai BACKGROUND: Chronic inflammation in adipose tissue together with obesity induces insulin resistance. Inhibitors of chronic inflammation in adipose tissue can be a potent candidate for the treatment of diabetes; however, only a few compounds have been discovered so far. The objective of this study was to find a novel inhibitor that can suppress the inflammatory response in adipose tissue and to elucidate the intracellular signaling mechanisms of the compound. METHODS: To find the active compounds, we established an assay system to evaluate the inhibition of induced MCP-1 production in adipocyte/macrophage coculture in a plant extract library. The active compound was isolated by performing high-performance liquid chromatography (HPLC) and was determined as 4β-hydroxywithanolide E (4βHWE) by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) spectral analyses. The effect of 4βHWE on inflammation in adipose tissue was assessed with adipocyte culture and db/db mice. RESULTS: During the screening process, Physalis pruinosa calyx extract was found to inhibit production of MCP-1 in coculture strongly. 4βHWE belongs to the withanolide family of compounds, and it has the strongest MCP-1 production inhibitory effect and lowest toxicity than any other withanolides in coculture. Its anti-inflammatory effect was partially dependent on the attenuation of NF-κB signaling in adipocyte. Moreover, in vivo experiments showed that the oral administration of 4βHWE to db/db mice resulted in the inhibition of macrophage invasion and cytokine expression in adipose tissue after 2 weeks of treatment; improved the plasma adiponectin, non-esterified fatty acids and MCP-1 concentrations; and increased glucose tolerance after 3 to 4 weeks of treatment. CONCLUSIONS: These results suggest that 4βHWE has anti-inflammatory effect via inhibition of NF-κB activation in adipocyte. Moreover, the attenuation of inflammation in adipocyte has an effect on the inhibition of macrophage accumulation in obese adipose tissue. Consequently, 4βHWE improves impaired glucose tolerance. Thus, 4βHWE is a useful natural anti-inflammatory compound to attenuate progression of diabetes and obesity. International Journal of Obesity (2014) 38, 1432–1439; doi:10.1038/ijo.2014.33 Keywords: anti-inflammation; adipose tissue; glucose metabolism; withanolide; inflammatory cytokines INTRODUCTION system. Consequently, we screened a library of extracts and Obesity and systemic insulin resistance are the primary causes of focused on Physalis pruinosa extract. type 2 diabetes. Chronic low-grade inflammation is thought to Recently, many physalins that are classified as secosteroids have account for the acquisition of insulin resistance.1 In both humans been isolated from Physalis alkekengi, and these compounds 9,10 and rodents, monocyte chemoattractant protein-1 (MCP-1) secre- have been reported to have anti-inflammatory and anticancer 11 tion from hypertrophic adipocytes induces monocyte infiltration effects. However, the pharmacological activity of P. pruinosa or into adipose tissue, and this phenomenon causes the secretion of its active components has almost never been reported. proinflammatory mediators from invasive macrophages and In contrast, some natural steroids and withanolides from plants adipocytes in adipose tissue.2,3 Accordingly, the basis of systemic or insects have been reported to improve impaired glucose 12–15 insulin resistance is thought to be the enhancement of tolerance and/or obesity. In the present study, plant sterols proinflammatory cytokine production in adipose tissue.3 with withanolide skeletons were isolated from P. pruinosa calyxes. Moreover, it has been reported that the inhibition of the secretion Among the withanolides, moleculer mechanisms of its anticancer of proinflammatory cytokines (for example, tumor necrosis effect of Withaferin A (WA) are extensively well studied. The factor-α (TNF-α) and MCP-1) or cytokines downstream in the inhibition of the nuclear factor-κB (NF-κB) signaling pathway signaling pathway and the inhibition of macrophage chemotaxis through the overphosphorylation of IκB kinase (IKK)-β16 and the have the ability to alleviate insulin resistance.4–8 upregulation of prostate apoptosis response-4 gene expression is To find natural compounds that can suppress the secretion of considered as the key mechanism of WA's action.17 In addition, MCP-1 in adipose tissue, we established a microplate-based WA has anticancer effects resulting from direct interactions with high-throughput adipocyte and macrophage coculture assay cellular structural proteins such as annexin II18 and vimentin.19 Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., Kawasaki-Ku, Kawasaki, Japan. Correspondence: Dr M Bannai, Frontier Research Laboratories, Institute for Innovation, Ajinomoto Co., Inc., 1-1 Suzuki-Cho, Kawasaki-Ku, Kawasaki 210- 8681, Japan. E-mail: [email protected] Received 28 August 2013; revised 27 January 2014; accepted 27 January 2014; accepted article preview online 25 February 2014; advance online publication, 25 March 2014 4βHWE improves inflammatory signaling in fat T Takimoto et al 1433 In this study, the P. pruinosa calyx was found to improve glucose (R&D Systems, Minneapolis, MN, USA) or RAW264.7 cells for 5, 15 or 30 min. metabolism based on the anti-inflammatory effect that this plant The cells were then collected with sample buffer, containing 10% glycerol, extract exhibited in the macrophage and adipocyte coculture phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and protease assay. We attempted to isolate the active compound from the inhibitor cocktail (Nacalai Tesque) and stored in a freezer at − 80 °C. P. pruinosa calyx and to elucidate its chemical structure. We also Nucleotides were degraded with TurboNuclease (Accelagen, San Diego, fl CA, USA), and the lysate was loaded onto a 10.5% SDS-polyacrylamide gel, studied the effect of the isolated compound on the in ammatory and electrophoresis was performed. The proteins were subsequently response in adipose tissue and glucose tolerance in diabetic mice. transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, We attempted to elucidate the intercellular signaling mechanisms CA, USA) with a Mini Trans-Blot Cell (Bio-Rad Laboratories). Immunoblot- of the anti-inflammatory effects of the active compound. ting was carried out with the following primary antibodies: anti-phospho- transforming growth factor-β-activated kinase (TAK) 1 antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA), anti-phospho-IKKα/β anti- MATERIALS AND METHODS body (1:1000; Cell Signaling Technology), anti-IκB-α antibody (1:1000; Cell Signaling Technology) and anti-α-tubulin antibody (1:1000; Cell Signaling Plant extract library Technology). Available edible plants including vegetables, fruits, spices and herbs were To study the effect of 4βHWE on NF-κB translocation to the nucleus, purchased. The 1030 types of divided plant parts were freeze-dried and nuclei were extracted from adipocytes that had been stimulated with then crushed into powders with a food processor. The powders were 10 ng ml − 1 recombinant mouse TNF-α for 30 min after a 10-min incubation extracted with methanol (that is, 1 g of each powder was soaked in 40 ml with or without 4βHWE. The cells were washed with phosphate-buffered of methanol), the solid phase was removed by filtration, and the liquid saline (PBS) and homogenized in lysis buffer with a Dounce homogenizer phase was dried at 30 °C with an evaporator. The dried extract was and centrifuged. Nuclear extraction buffer was added to the pellet, and the − 1 dissolved in dimethylsulfoxide (DMSO) to a concentration of 100 mg ml mixture was placed on ice for 45 min. The supernatant was obtained by and stored at − 80 °C. centrifugation and stored at − 80 °C. Each protein was loaded onto a 10.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. κ TNF-α assay in macrophages Immunoblotting was carried out with the NF- B p50 antibody and Histone H1 antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). RAW264.7 cells (a mouse macrophage cell line; #TIB-71; ATCC, Rockville, Histone H1 was used as a loading control. MD, USA) were cultured overnight in RPMI 1640 (Invitrogen, Carlsbad, CA, To detect the primary antibodies, anti-rabbit IgG conjugated to USA) supplemented with 0.2% FBS. Plant extracts were added to the horseradish peroxidase (1:10 000 GE Healthcare, Little Chalfont, UK) was μ − 1 culture medium at concentrations of 3, 10, 30 and 100 gml , and then used as a secondary antibody. Immunoblots were visualized by using an μ 400 M BSA-conjugated palmitate was added to stimulate the cells. After LAS-3000 imaging system (Fujifilm Corp., Tokyo, Japan) with the ECL Plus α 24 h, the medium was collected, and the TNF- level was measured using System (GE Healthcare) according to the manufacturer’s guidelines. an ELISA kit (Thermo Fisher Scientific, Tewksbury, MA, USA). Luciferase assay for NF-κB transcriptional activity MCP-1 assay using the adipocyte/macrophage coculture system CV-1 cells (African green monkey kidney cells)