Hydroxywithanolide E Isolated from Physalis Pruinosa Calyx Decreases Inﬂammatory Responses by Inhibiting the NF-Κb Signaling in Diabetic Mouse Adipose Tissue

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) were transfected with 3T3-L1 cells (ATCC) were differentiated into mature adipocytes by using a the PathDetect NF-κB Cis-Reporting System (Stratagene, La Jolla, CA, USA) 20 conventional protocol. The medium was then removed, each plant by the FuGENE6 Transfection Reagent (Roche, Mannheim, Germany). The − 1 extract was added to RAW264.7 cells, and the two cell lines were transfected cells were incubated with 10 μM 4βHWE and 10 ng ml cocultured in DMEM supplemented with 0.5% BSA and 0.1% DMSO as a TNF-α for 24 h, and the luciferase activity was detected with the ONE-Glo vehicle. After 24 h, the medium was collected, and the level of MCP-1 was Luciferase Assay System (Promega, Madison, WI, USA) according to the determined using an ELISA kit (BD Biosciences-Pharmingen, San Jose, CA, manufacturer’s guidelines. USA). The viability of the cells was measured with the Cell Counting Kit-8 (Dojindo, Osaka, Japan) according to the manufacturer’s guidelines. RNA extraction and real-time RT–PCR fi β Total RNA was extracted from cells or tissues and reverse transcribed into The puri cation of 4 -hydroxywithanolide E from P. pruinosa calyx complementary DNA (cDNA). Real-time RT–PCR analysis was subsequently extract performed with the Thermal Cycler Dice Real Time System (Takara Bio, P. pruinosa calyx was obtained from the Akita prefecture in the northern Shiga, Japan). Each reaction tube contained template, 0.5 μM each primer part of Japan. Using the extraction method described above, 10.12 g of and SYBR Premix Ex Taq II (Takara Bio). The following real-time PCR dried material was obtained from 340 g of P. pruinosa calyx. The dried conditions were used: 40 cycles of 94 °C for 5 s, 60 °C for 45 s and 95 °C for material was extracted with methanol, and the extract was separated by 30 s. The primer sequences are shown in Supplementary Table 1. column chromatography using Silica gel 60 N (Kanto Chemical, Tokyo, The results were analyzed using the 2-ΔΔCt method (ABI User Bulletin 2) Japan). The fifth and sixth fractions exhibited anti-inflammatory activity, and are presented as the ratio of each mRNA to the 18S rRNA to correct for evaluated by using the MCP-1 assay. These fractions were separated again variations in the quantities of the template DNA. by Inertsil ODS-3 250 × 50 mm ID (GL Sciences, Torrance, CA, USA), and the purified active compound was analyzed using a total ion chromatogram collected with a photodiode array and LC/MS (Quattro Micro; Waters Corp., Animal experiment Milford, MA, USA). The compound was identified as 4β-hydroxywithanolide The experimental protocol was reviewed and approved by the Animal Care E(4βHWE; molecular weight 502.6) by NMR (AVANCE 400; Bruker BioSpin Committee of Ajinomoto. Five-week-old male db/db and db/+ mice were Corp., Billerica, MA, USA) and LC/MS. From 10.12 g dried P. pruinosa calyx purchased from the Charles River Laboratories (Yokohama, Japan) and methanol extract (PME), we obtained about 240 mg 4βHWE and these were tamed for 1 week under the experimental conditions. The food and compounds were used to evaluate its effects in further experiments. drinking water were provided ad libitum. The mice were divided into 4 groups: db/db mice administered with vehicle (0.5% carboxymethyl cellulose; Wako Pure Chemical Industries, Osaka, Japan), db/db mice The effect of 4βHWE on macrophage and adipocyte cytotoxicity administered with 10 mg kg − 1 body weight of 4βHWE, db/db mice − 4βHWE or the vehicle (0.5% BSA and 0.1% DMSO in DMEM) was added into administered with 30 mg kg 1 body weight of 4βHWE and db/+ mature 3T3-L1 or RAW264.7 cells. An equal volume of coculture medium mice administered with vehicle. Mice were orally administered once per was added simultaneously. After 24 h, the cell viability was measured as day for 2 or 4 weeks. described above. To quantify the mRNA expression levels of proinflammatory cytokines (TNF-α, MCP-1 and interleukin (IL)-6) and macrophage markers (F4/80 and CD68), the mice were killed and epididymal adipose tissue was collected Immunoblot analysis after 2 weeks of treatment. A portion of the epididymal adipose tissue was Mature 3T3-L1 cells were incubated with 4βHWE or withaferin A fixed with neutral-buffered formalin for immunohistological analysis. (WA; Alexis Lausanne, Switzerland) for 10 min. After incubation, Glucose tolerance tests were performed after 3 weeks of treatment. In these cells were stimulated with 10 ng ml − 1 recombinant mouse TNF-α brief, mice were orally administered 0.75 g kg − 1 body weight of glucose,

© 2014 Macmillan Publishers Limited International Journal of Obesity (2014) 1432 – 1439 4βHWE improves inflammatory signaling in fat T Takimoto et al 1434 and blood was collected before and 15, 30, 60 and 120 min after the administration. Blood glucose levels were measured with a glucose analyzer (ARKRAY, Kyoto, Japan). After 4 weeks of treatment, blood samples were collected, and the expression of mRNA in the adipose tissue was measured using the real-time RT–PCR method described above. The plasma MCP-1 and adiponectin concentrations were measured with an ELISA kit (BD Biosciences-Pharmingen and Otsuka Pharmaceutical, Tokushima, Japan, respectively). Plasma-free fatty acids were measured with the Wako NEFA C test kit (Wako Pure Chemical Industries). Macrophages in the adipose tissue were detected by immunohisto- chemistry. In brief, tissues were embedded in paraffin and cut into 5-μm sections. An anti-mouse macrophage antibody (F4/80; BMA Biomedicals, Rheinstrasse, Switzerland) was used as the primary antibody (1:200), and immunoreactivity was detected with the avidin–biotin complex (VECTAS- TAIN ABC; Vector Laboratories, Burlingame, CA, USA). The tissue was subsequently counterstained with hematoxylin. The number of F4/80- positive cells was counted under a microscope in a 0.0625 mm2 field of view. More than 50 serial fields were examined, and the data were expressed as cells per mm2 of adipose tissue.

Statistical analyses The data are presented as the mean ± s.e. of the mean. For experiments with two factors, for example, concentration and drugs (Figures 2 and 3d–f) or time and treatment groups (Figure 5a), significant differences between groups and doses/time points were identified by a two-way ANOVA followed by Bonferroni’s multiple comparisons test. For experiments with one factor, for example, drug dose, significant differences were identified using a one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test.

RESULTS We screened 1030 plant extracts from the plant library using the adipocyte/macrophage coculture assay, and 10 plant extracts exhibited a strong inhibitory effect on MCP-1 production. These 10 plant extracts were derived from P. pruinosa, Rabdosia japonica, Coptis japonica Makino, Zingiber officinale Roscoe, Nuphar japonicum De Candolle, Ephedra sinica Stapf, Withania somnifera, Curcuma longa, Lycoris radiate, and Rosmarinus officinalis L. From the potent candidates, we focused on the extract of P. pruinosa because this extract had one of the strongest activities on MCP-1 production without inducing cytotoxicity. The PME inhibited TNF-α production from macrophages that was induced by palmitate in a dose-dependent manner (Figure 1a). In addition, PME also inhibited MCP-1 production in the adipocyte/macrophage coculture system in a semi-dose- dependent manner without cytotoxicity in the range from 0 to 100 μgml− 1 (Figures 1b and c). These anti-inflammatory effects were exerted without any cytotoxicity. The active compound in the PME was identified as Figure 1. The effects of P. pruinosa methanol extract (PME) on the 4β-hydroxywithanolide E (4βHWE; Figure 2a) with a photodiode TNF-α production stimulated by palmitate (a), on the MCP-1 array and by NMR and LC/MS spectral analysis. production induced by the RAW264.7 (macrophage) and 3T3-L1 The inhibitory effects of the withanolides on MCP-1 secretion (adipocyte) coculture (b) and on cytotoxicity in RAW264.7 and 3T3- L1 coculture (c). Values are presented as the mean ± s.e.m. of three were evaluated using the adipocyte/macrophage coculture assay. o o The results are shown in Figure 2b, and the half maximal inhibitory separate wells. * represents P 0.05 and ** represents P 0.01; these asterisks indicate statistically significant differences relative to concentrations of MCP-1 secretion were 1.59 ± 0.28 μM for 4βHWE, μ μ β the value obtained under stimulated conditions without PME, as 5.92 ± 0.82 M for WA, 10.48 ± 1.8 M for 3-methoxy-4 HWE and identified through a one-way ANOVA with Bonferroni’s test. >30 μM for withanolide B. Neither withanolide S nor withaperuvin C, which are analogs of the withanolides purified from PME, showed any anti-inflammatory effects at concentrations up to However, both 4βHWE and WA decreased the viability of 10 μM (data not shown). macrophages in a dose-dependent manner. The negative effect The effects of 4βHWE and WA on cell viability were examined of 4βHWE on macrophage viability was significantly weaker than using 3T3-L1 adipocytes (Figure 2c), RAW264.7 macrophages the negative effect of WA (Figure 2d). (Figure 2d) and adipocyte/macrophage coculture (Figure 2e) with To elucidate the anti-inflammatory mechanisms of 4βHWE, concentrations ranging from 0.1 to 30 μM. Neither 4βHWE nor WA TNF-α was used to stimulate changes in the intracellular signaling affected the viability of adipocytes at concentrations between 0.1 of adipocytes. After 5 min of TNF-α stimulation, the phosphorylation and 10 μM. However, WA but not 4βHWE reduced the viability of levels of TAK-1 and IKKβ were increased, and the IκBα protein level adipocytes when added at concentrations of 30 μM (Figure 2c). In was decreased in 3T3-L1 adipocytes (Figure 3a). In particular, the coculture, the effects of these compounds on cell viability TAK-1 phosphorylation increased and IκBα level decreased were similar to their effects on adipocytes alone (Figure 2e). gradually until after 30 min of TNF-α stimulation. The addition of

Figure 2. The chemical structure of 4βHWE (a), the effect of withanolides on the production of MCP-1 induced by the adipocyte and macrophage coculture (b) and the effect of withanolides on the viability of 3T3-L1 adipocytes (c), RAW264.7 macrophages (d) and cocultured cells (e). Values are presented as the mean ± s.e.m. of three well determinations. * represents Po0.001 and ** represents Po0.0001; these asterisks indicate statistically significant differences when relative to the treatment, as identified through a two-way ANOVA with Bonferroni’s test. NS, not significant. Figure 3. The effect of 4βHWE on the NF-κB signaling pathway after upregulation by TNF-α (10 ng ml − 1) in 3T3-L1 adipocytes. Panel a shows the time course of intracellular signal transduction with or 10 μM 4βHWE to the medium inhibited the inflammatory changes without 10 μM 4βHWE in adipocytes. Panel b shows intranuclear in intracellular signaling in the adipocytes (Figure 3a). NF-κB in adipocytes after 30 min of TNF-α stimulation with or Moreover, the level of NF-κB was increased in the nuclei of the without 10 μM 4bHWE. Panel c shows the effect of 10 μM 4βHWE on adipocytes after 30 minutes of TNF-α stimulation. The addition of NF-κB transcriptional activity. The effect of 5 μM 4βHWE on the μ β mRNA expression of MCP-1 (d), TNF-α (e), and IL-6 (f) in adipocytes 10 M 4 HWE to the medium inhibited the increase in intranuclear α ± NF-κB induced by TNF-α (Figure 3b). stimulated by TNF- is shown. Values are presented as the mean s. e.m. of three well determinations. In panel c, *** represents CV-1 cells that were transfected with a vector that contained o fi fi κ P 0.001 and indicates statistically signi cant differences when ve repeats of the NF- B- binding site sequence upstream of the compared with the value obtained in the stimulated condition without luciferase protein coding sequence were used to elucidate the 4βHWE, as identified through a one-way ANOVA with Bonferroni’stest. effect of 4βHWE on the transcriptional activity of NF-κB. As shown In panels d–f, ** represents Po0.01 and *** represents Po0.001; in Figure 3c, 4βHWE significantly inhibited the upregulation of these asterisks indicate statistically significant differences relative to luciferase activity by TNF-α. the treatment, as identified through a two-way ANOVA. Next, the effect of 4βHWE on the mRNA expression of inflammatory cytokines induced by TNF-α was examined in − adipocytes. MCP-1 mRNA expression increased approximately A group treated with 10 mg kg 1 of 4βHWE exhibited a 30–40 times after 2, 4 and 6 h of TNF-α stimulation, and 4βHWE significant increase in body weight compared with the vehicle − significantly inhibited this upregulation of MCP-1 mRNA expres- group; however, at a higher dose (30 mg kg 1 of 4βHWE), there sion (Figure 3d). The TNF-α mRNA level increased approximately were no significant differences (Supplementary Table 2). 3000-fold after a 2-h stimulation, and this elevation was The weight of subcutaneous, epididymal and mesenteric adipose attenuated by 4βHWE (Figure 3e). The IL-6 mRNA level also tissues were not significantly different among the groups. There increased twofold after a 2-h stimulation, and 4βHWE attenuated were no significant differences in the number of white blood this increase in expression (Figure 3f). These suppressions were cells (WBC), glutamate oxaloacetate transferase (GOT), glutamate similar to the effects of WA after a 2-h stimulation (Figures 3d–f). pyruvate transaminase (GPT) and creatinine concentrations To evaluate the effects of 4βHWE on the inflammatory response among the groups (Supplementary Table 3). and glucose tolerance in diabetic mice, 4βHWE (10 or 30 mg kg − 1 After 2 weeks of 4βHWE administration, F4/80-positive cells, body weight) was orally administered daily to db/db mice for which were used as indicator of macrophages, infiltrated into the 4 weeks. epididymal adipose tissue of db/db mice; the level of macrophage

Figure 4. The effect of the oral administration of 4βHWE (10 or 30 mg kg − 1 body weight per day) for 2 weeks on the inflammatory responses in the epididymal adipose tissue of db/db mice. Representative examples of tissue sections in which the macrophages were stained with an anti-F4/80 antibody and the nucleus was counterstained with hematoxylin are shown in panels (a–d). The effect of 4βHWE on the number of F4/80-positive cells (e) and on the mRNA expression of macrophage markers (F4/80 (f) and CD68 (g)) and inflammatory cytokines (TNF-α (h), MCP-1 (i) and IL-6 (j)) in adipose tissue are shown. The scale bar indicates 50 μm in panels a–d, and the arrowhead indicates macrophages. The values are presented as the mean ± s.e.m. of 5 and 8 individual determinations in panels e and f–j, respectively. * represents Po0.05 and ** represents Po0.01; the asterisks indicate statistically significant decreases relative to vehicle-treated db/db mice, as identified by a one-way ANOVA with Bonferroni’s test.

infiltration was reduced by 4βHWE (Figures 4a–d). Figure 4e shows proinflammatory cytokines (MCP-1, TNF-α and IL-6) was simulta- the effect of 4βHWE on the number of macrophages in adipose neously suppressed by 4βHWE in a dose-dependent manner tissue; 4βHWE reduced the infiltration of macrophages in a (Figures 4h–j). dose-dependent manner. In addition, the mRNA expression After 3 weeks of 4βHWE administration, the mice were levels of macrophage-specific cellular membrane proteins (F4/80 subjected to a glucose tolerance test. db/db Mice exhibited and CD68) in adipose tissue were also reduced by 4βHWE increased blood glucose levels, and this elevation was (Figures 4f and g). The mRNA expression of the obesity-induced attenuated by treatment with 30 mg kg − 1 of 4βHWE for 3 weeks

Figure 5. The effect of the oral administration of 4βHWE (10 or 30 mg kg − 1 body weight per day) for 3 weeks on glucose availability (a), area under the curve (AUC) of glucose concentration (b)indb/db mice. The effect of oral administration of 4βHWE for 4 weeks on basal plasma glucose concentration (c), and plasma insulin concentration (d)indb/db mice. The values are presented as the mean ± s.e.m. of 8–10 individual determinations. In panel a, * represents Po0.05, which indicates significant decreases relative to vehicle-treated db/db mice, as identified through a two-way ANOVA with Bonferroni’s test. In panels b and c, * represents Po0.05 and *** represents Po0.001; the asterisks indicate statistically significant decreases relative to vehicle-treated db/db mice, as identified through a one-way ANOVA with Bonferroni’s test.

Figure 6. The effect of the oral administration of 4βHWE (10 or 30 mg kg − 1 body weight per day) for 4 weeks on the plasma concentrations of adiponectin (a), MCP-1 (b) and free fatty acids (c)indb/db mice. The values are presented as the mean ± s.e.m. of 10 individual determinations. * represents Po0.05 and ** represents Po0.01; the asterisks indicate statistically signiﬁcant differences relative to vehicle-treated db/db mice, as identiﬁed through a one-way ANOVA with Bonferroni’s test.

(Figures 5a and b). In addition, the administration of 30 mg kg − 1 From those, P. pruinosa calyx extract was identified to possess of 4βHWE for 4 weeks significantly decreased the blood glucose strong inhibitory effect on MCP-1 production induced in levels in db/db mice relative to the levels in db/db mice that were adipocyte/macrophage coculture assay. We then attempted to treated with the vehicle alone (Figure 5c). The plasma insulin isolate the possible active compound from P. pruinosa calyx and concentrations did not change at this time point (Figure 5d). identified it as 4βHWE, which is a member of the withanolide The treatment of 30 mg kg − 1 of 4βHWE for 4 weeks significantly family of compounds. 4βHWE had a stronger anti-inflammatory increased plasma adiponectin and decreased MCP-1 levels effect and lower toxicity than any other withanolides and that its compared with the vehicle groups. (Figures 6a and b). Also, the anti-inflammatory effect was dependent on the attenuation of concentration of plasma-free fatty acids decreased significantly NF-κB signaling. In vivo experiments showed that the oral (Figure 6c). administration of 4βHWE to obese db/db mice inhibited the inflammatory responses in adipose tissue and improved impaired glucose tolerance. DISCUSSION First, we verified that PME attenuated palmitate-induced In this study, we screened 1030 plant extracts to identify potent production of TNF-α in macrophages and MCP-1 in the macro- compounds with the ability to inhibit the diabetes progression. phage/adipocyte coculture system (Figures 1a and b). Cocultures

© 2014 Macmillan Publishers Limited International Journal of Obesity (2014) 1432 – 1439 4βHWE improves inflammatory signaling in fat T Takimoto et al 1438 of 3T3-L1 adipocytes and RAW264.7 macrophages are used as mouse obesity models, such as high-fat-diet-induced obese mice, in vitro models of inflammatory changes in obese animals.21,22 db/db and ob/ob mice. In these studies, the inflammatory These results indicate that PME contains anti-inflammatory responses in adipose tissue were alleviated when the secretion compounds that inhibit macrophage activation and infiltration of adipokines (for example, TNF-α, MCP-1 and FFA) decreased and into adipose tissue caused by adipocyte hypertrophy. Physalis adiponectin level increased. As a result, systemic insulin sensitivity extracts have been reported to regulate the estrus cycle,23 the might be increased and/or glucose tolerance was improved. activity of glucose-6-P dehydrogenase in hepatocytes,24 induce Thus, we attempted to examine the effects of 4βHWE on the cancer cell apotosis,25,26 histopathological changes27 and inflam- inflammatory responses in the adipose tissue of an obese mouse matory changes.28–30 In addition, the anti-proliferative effect on model. The oral administration of 4βHWE (30 mg kg − 1)todb/db HTLV-1-infected T-cell lines has been reported only with the mice for 2 weeks decreased the number of macrophages, the extract of P. pruinosa.31 Therefore, we attempted to isolate and mRNA expression of macrophage-specific membrane antigens in identify anti-inflammatory compounds present in PME and abdominal adipose tissue (Figures 4a–g) and the expression of identified 4βHWE. 4βHWE is a withanolide, a family that has more proinflammatory cytokines (Figures 4h–j). The inhibition of the than 60 types. Most of the known withanolides have been isolated expression of proinflammatory cytokines by 4βHWE might be from Tubocapsicum anomalum or Withania somnifera Dunal not dependent on the suppression of macrophage recruitment in from the genus physalis, and these compounds have been obese animals’ adipose tissue. This inhibition of macrophage reported to possess variety of physiological activities in vivo infiltration effect might be due to inhibition of obesity-induced and/or in vitro, including anticancer,32,33 anti-inflammatory16,34 inflammatory cytokine production from adipocyte as seen in and neuroprotective 35,36 activities. 4βHWE was originally found in in vitro experiments. Moreover, administration of 30 mg kg − 1 Physalis peruviana L.37 and has been intensively studied as an 4βHWE for 3 or 4 weeks suggested improvement of impaired inhibitor of cancer cell proliferation in vitro.38,39 The present study glucose tolerance (Figures 5a and b), which involved an increase is the first report of the isolation of 4βHWE from P. pruinosa, and in plasma adiponectin levels and decrease in the plasma MCP-1 our experiments suggested a novel physiological function of and free fatty acid levels in db/db mice (Figures 6a–c). These 4βHWE as a compound that has anti-inflammatory effects that effects also may be dependent on the suppression of the involve an increase in glucose tolerance. inflammatory responses in the adipose tissue. 4βHWE exhibited the strongest inhibition of MCP-1 production, Recently, the gut microbiome has been recognized as a key with the least cytotoxicity than the other four withanolides that regulator of metabolic health in humans.46,47 Because it is not were isolated during the isolation of the active compound clear whether 4βHWE altered the microbiome in this study, further 38 (Figures 2b–e). Yen et al. reported that 2, 10 and 20 μM 4βHWE studies on the effects of 4βHWE on the microbiome are required. significantly induced DNA damage in a dose-dependent manner We investigated basal glucose and insulin concentrations after a in a human lung cancer cell line (H1299). However, in our 4-week 4βHWE administration (Figures 5c and d). The plasma experiments, 30 μM 4βHWE was not toxic to a mature adipocyte glucose concentrations decreased in a dose-dependent manner; cell line (3T3-L1; Figures 1c and 2c), whereas it showed toxic however, the insulin concentrations did not change significantly. effects at 3 μM in a macrophage cell line (RAW264.7; Figure 2d). These results suggest that rather than acting on insulin secretion, These results suggest that the toxic effects of 4βHWE might differ 4βHWE affects insulin sensitivity. To confirm the effects on insulin according to cell type. To elucidate the mechanisms of the anti- sensitivity, an insulin tolerance test was performed. 4βHWE slightly inflammatory effects of 4βHWE, NF-κB signal transduction was increased glucose clearance, but the effect was not significant evaluated in TNF-α-stimulated 3T3-L1 cells. 4βHWE significantly (data not shown). Although the mechanism has not been inhibited TNF-α-induced proinflammatory responses, including completely elucidated, long-term 4βHWE administration might the induction of TAK-1, IKKβ phosphorylation and the degradation produce qualitative changes in adipose tissue rather than directly of IκBα (Figure 3a). Moreover, 4βHWE suppressed the translocation influencing glucose uptake and cellular signaling. 4βHWE of NF-κB into the nucleus and thus inhibited the transcriptional inhibited the inflammatory response in adipose tissue and may activity of NF-κB (Figures 3b and c). The mRNA expression of have the potential to improve impaired glucose tolerance in db/db proinflammatory cytokines such as MCP-1, TNF-α and IL-6 was mice. 4βHWE is expected to prevent the development of glucose decreased by 4βHWE (Figures 3d–f). Generally, mRNA expression metabolism impairment in obese individuals. of these cytokines is upregulated by the activation of NF-κB and In conclusion, this study showed that 4βHWE could have the acceleration of the inflammatory response.40–42 Thus, the anti- greater beneficial anti-inflammatory effects than other with- inflammatory effects of 4βHWE could possibly be caused by the anolides, which are known to have a wide range of physiological inhibition of NF-κB signaling. Although WA has been reported to activities. 4βHWE may be useful as a natural anti-inflammatory inhibit the IKKβ activation through the induction of IKKβ compound for the prevention of type 2 diabetes and metabolic overphosphorylation,16 4βHWE inhibited the IKKβ activation syndrome. through the suppression of IKKβ phosphorylation in adipocytes (Figure 3a). The diverse regulation of IKKβ phosphorylation observed in this study might be caused by the differences between the chemical structures of 4βHWE and WA. In the calculated stable structural form of 4βHWE, the flat surface of the CONFLICT OF INTEREST fl – lactone ring is skewed toward the at surfaces of the A D rings T Takimoto, Y Kanbayashi, T Toyoda, Y Adachi, C Furuta, K Suzuki, T Miwa and (PubChem Compound Identifier (CID) 73621). However, in the M Bannai are employees of Ajinomoto. calculated stable structural form of WA, both the flat surface of the lactone ring and the surfaces of the A–D rings lie in the same plane (CID 265237). Thus, these differences in the stable chemical structure might be the cause of the distinct mechanisms of action of these two compounds. Taken together, these results led us to ACKNOWLEDGEMENTS speculate that the anti-inflammatory mechanisms or molecular β We thank Hiroaki Kisaka and Ryuji Sugiyama for supplying the plant extract library targets of 4 HWE and WA are different. and the Physalis calyx from Kamikoani, Shin Harumatsu for providing technical fl 43 β Anti-in ammatory compounds, including salicylates, IKK assistance and Reika Nakagawa for the screening and identification of active fractions 44 45 12–15 inhibitors, curcumin and natural steroids or triterpenoids, with the coculture assay. We also thank Naoko Akimoto for her assistance with have been reported to improve impaired glucose tolerance in 4βHWE purification.

International Journal of Obesity (2014) 1432 – 1439 © 2014 Macmillan Publishers Limited 4βHWE improves inflammatory signaling in fat T Takimoto et al 1439 REFERENCES 24 Vessal M, Mostafavi-Pour Z, Kooshesh F. Age and sex dependence of the effects of 1 Odegaard JI, Chawla A. Mechanisms of macrophage activation in an aqueous extract of Physalis alkekengi fruits on rat hepatic glucose 6-P dehy- obesity-induced insulin resistance. Nat Clin Pract Endocrinol Metab 2008; 4: drogenase activity. Comp Biochem Physiol Part B, Biochem Mol Biol 1995; 111: – 619–626. 675 680. 2 Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. 25 Wu SJ, Ng LT, Chen CH, Lin DL, Wang SS, Lin CC. Antihepatoma activity of Physalis Obesity is associated with macrophage accumulation in adipose tissue. J Clin angulata and P. peruviana extracts and their effects on apoptosis in human Hep Investig 2003; 112: 1796–1808. G2 cells. Life Sci 2004; 74: 2061–2073. 3 Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ et al. Chronic inflammation in fat 26 Quispe-Mauricio A, Callacondo D, Rojas J, Zavala D, Posso M, Vaisberg A. plays a crucial role in the development of obesity-related insulin resistance. J Clin [Cytotoxic effect of physalis peruviana in cell culture of colorectal and prostate Investig 2003; 112: 1821–1830. cancer and chronic myeloid leukemia]. Revista de gastroenterologia del Peru 2009; 4 Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM et al. IKK-beta links 29:239–246. inflammation to obesity-induced insulin resistance. Nat Med 2005; 11:191–198. 27 Arun M, Asha VV. Preliminary studies on antihepatotoxic effect of Physalis 5 De Taeye BM, Novitskaya T, McGuinness OP, Gleaves L, Medda M, Covington JW peruviana Linn. (Solanaceae) against carbon tetrachloride induced acute liver et al. Macrophage TNF-alpha contributes to insulin resistance and hepatic injury in rats. J Ethnopharmacol 2007; 111:110–114. steatosis in diet-induced obesity. Am J Physiol Endocrinol Metab 2007; 293: 28 Bastos GN, Silveira AJ, Salgado CG, Picanco-Diniz DL, do Nascimento JL. Physalis fl E713–E725. angulata extract exerts anti-in ammatory effects in rats by inhibiting different 6 Solinas G, Vilcu C, Neels JG, Bandyopadhyay GK, Luo JL, Naugler W et al. JNK1 in pathways. J Ethnopharmacol 2008; 118:246–251. hematopoietically derived cells contributes to diet-induced inflammation and 29 Martinez W, Ospina LF, Granados D, Delgado G. In vitro studies on the relationship insulin resistance without affecting obesity. Cell Metab 2007; 6: 386–397. between the anti-inflammatory activity of Physalis peruviana extracts and the 7 Verschuren L, Kooistra T, Bernhagen J, Voshol PJ, Ouwens DM, van Erk M et al. phagocytic process. Immunopharmacol Immunotoxicol 2010; 32:63–73. MIF deficiency reduces chronic inflammation in white adipose tissue and impairs 30 Kang H, Kwon SR, Choi HY.. Inhibitory effect of Physalis alkekengi L. var. franchetii the development of insulin resistance, glucose intolerance, and associated extract and its chloroform fraction on LPS or LPS/IFN-gamma-stimulated inflam- 135 atherosclerotic disease. Circ Res 2009; 105:99–107. matory response in peritoneal macrophages. J Ethnopharmacol 2011; : 8 Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R et al. MCP-1 contributes 95–101. to macrophage infiltration into adipose tissue, insulin resistance, and hepatic 31 Nakano D, Ishitsuka K, Hatsuse T, Tsuchihashi R, Okawa M, Okabe H et al. steatosis in obesity. J Clin Investig 2006; 116: 1494–1505. Screening of promising chemotherapeutic candidates against human adult 9 Vieira AT, Pinho V, Lepsch LB, Scavone C, Ribeiro IM, Tomassini T et al. T-cell leukemia/lymphoma from plants: active principles from Physalis pruinosa Mechanisms of the anti-inflammatory effects of the natural secosteroids physalins and structure-activity relationships with withanolides. J Nat Med 2011; 65: – in a model of intestinal ischaemia and reperfusion injury. Br J Pharmacol 2005; 559 567. 146: 244–251. 32 Gunasekera SP, Cordell GA, Farnsworth NR. Plant Anticancer Agents XX.* 10 Jacobo-Herrera NJ, Bremner P, Marquez N, Gupta MP, Gibbons S, Munoz E et al. Constituents of Nicandra physalodes. Planta Med 1981; 43:389–391. Physalins from Witheringia solanacea as modulators of the NF-kappaB cascade. 33 Cordero CP, Morantes SJ, Paez A, Rincon J, Aristizabal FA. Cytotoxicity of J Nat Products 2006; 69: 328–331. withanolides isolated from Acnistus arborescens. Fitoterapia 2009; 80: 364–368. 11 Lee CC, Houghton P. Cytotoxicity of plants from Malaysia and Thailand used 34 Budhiraja RD, Sudhir S, Garg KN. Anti-inflammatory activity of 3 beta-hydroxy-2,3- traditionally to treat cancer. J Ethnopharmacol 2005; 100:237–243. dihydro-withanolide F. Planta Med 1984; 50:134–136. 12 Kizelsztein P, Govorko D, Komarnytsky S, Evans A, Wang Z, Cefalu WT et al. 35 Kuboyama T, Tohda C, Komatsu K. Neuritic regeneration and synaptic recon- 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced struction induced by withanolide A. Br J Pharmacol 2005; 144:961–971. obesity mice model. Am J Physiol Endocrinol Metab 2009; 296: E433–E439. 36 Kuboyama T, Tohda C, Zhao J, Nakamura N, Hattori M, Komatsu K. Axon- or 13 Maurya R, Akanksha Jayendra, Singh AB, Srivastava AK. Coagulanolide, dendrite-predominant outgrowth induced by constituents from Ashwagandha. a withanolide from Withania coagulans fruits and antihyperglycemic activity. Neuroreport 2002; 13: 1715–1720. Bio-org Med Chem Lett 2008; 18: 6534–6537. 37 Sakurai K, Ishii H, Kobayashi S, Iwao T. Isolation of 4 beta-hydroxywithanolide E, 24 14 Xu A, Wang H, Hoo RL, Sweeney G, Vanhoutte PM, Wang Y et al. Selective a new withanolide from Physalis peruviana L. Chem Pharm Bull 1976; : elevation of adiponectin production by the natural compounds derived from a 1403–1405. medicinal herb alleviates insulin resistance and glucose intolerance in 38 Yen CY, Chiu CC, Chang FR, Chen JY, Hwang CC, Hseu YC et al. obese mice. Endocrinology 2009; 150: 625–633. 4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits 15 Lu M, Patsouris D, Li P, Flores-Riveros J, Frincke JM, Watkins S et al. growth of human lung cancer cells through DNA damage, apoptosis and A new antidiabetic compound attenuates inflammation and insulin resistance in G2/M arrest. BMC cancer 2010; 10: 46. Zucker diabetic fatty rats. Am J Physiol Endocrinol Metab 2010; 298: E1036–E1048. 39 Hsieh PW, Huang ZY, Chen JH, Chang FR, Wu CC, Yang YL et al. Cytotoxic with- 70 – 16 Kaileh M, Vanden Berghe W, Heyerick A, Horion J, Piette J, Libert C et al. Withaferin anolides from Tubocapsicum anomalum. J Nat Products 2007; :747 753. a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with 40 Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K et al. NF-kappa B and potent inhibition of its kinase activity. J Biol Chem 2007; 282: 4253–4264. Sp1 regulate transcription of the human monocyte chemoattractant protein- 17 Srinivasan S, Ranga RS, Burikhanov R, Han SS, Chendil D. Par-4-dependent 1 gene. J Immunol 1994; 153: 2052–2063. apoptosis by the dietary compound withaferin A in prostate cancer cells. Cancer 41 Shakhov AN, Collart MA, Vassalli P, Nedospasov SA, Jongeneel CV. Kappa B-type Res 2007; 67:246–253. enhancers are involved in lipopolysaccharide-mediated transcriptional activation 18 Falsey RR, Marron MT, Gunaherath GM, Shirahatti N, Mahadevan D, Gunatilaka AA of the tumor necrosis factor alpha gene in primary macrophages. J Exp Med 1990; et al. Actin microfilament aggregation induced by withaferin A is mediated by 171:35–47. annexin II. Nat Chem Biol 2006; 2:33–38. 42 Libermann TA, Baltimore D. Activation of interleukin-6 gene expression through 19 Bargagna-Mohan P, Hamza A, Kim YE, Khuan Abby Ho Y, Mor-Vaknin N, the NF-kappa B transcription factor. Mol Cell Biol 1990; 10: 2327–2334. Wendschlag N et al. The tumor inhibitor and antiangiogenic agent withaferin A 43 Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M et al. Reversal of targets the intermediate filament protein vimentin. Chem Biol 2007; 14:623–634. obesity- and diet-induced insulin resistance with salicylates or targeted disruption 20 Giri S, Rattan R, Haq E, Khan M, Yasmin R, Won JS et al. AICAR inhibits adipocyte of Ikkbeta. Science 2001; 293: 1673–1677. differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity 44 Kamon J, Yamauchi T, Muto S, Takekawa S, Ito Y, Hada Y et al. A novel IKKbeta mice model. Nutrition Metab 2006; 3:31. inhibitor stimulates adiponectin levels and ameliorates obesity-linked insulin 21 Suganami T, Nishida J, Ogawa Y. A paracrine loop between adipocytes and resistance. Biochem Biophys Res Commun 2004; 323:242–248. macrophages aggravates inflammatory changes: role of free fatty acids and 45 Weisberg SP, Leibel R, Tortoriello DV. Dietary curcumin significantly improves tumor necrosis factor alpha. Arterioscler Thromb Vasc Biol 2005; 25: 2062–2068. obesity-associated inflammation and diabetes in mouse models of diabesity. 22 Toyoda T, Kamei Y, Kato H, Sugita S, Takeya M, Suganami T et al. Effect of Endocrinology 2008; 149: 3549–3558. peroxisome proliferator-activated receptor-alpha ligands in the interaction 46 Turnbaugh PJ, Backhed F, Fulton L, Gordon JI. Diet-induced obesity is linked to between adipocytes and macrophages in obese adipose tissue. Obesity 2008; 16: marked but reversible alterations in the mouse distal gut microbiome. Cell Host 1199–1207. Microbe 2008; 3: 213–223. 23 Vessal M, Mehrani HA, Omrani GH. Effects of an aqueous extract of Physalis 47 Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM, Cusack S et al. Gut alkekengi fruit on estrus cycle, reproduction and uterine creatine kinase microbiota composition correlates with diet and health in the elderly. Nature BB-isozyme in rats. J Ethnopharmacol 1991; 34:69–78. 2012; 488: 178–184.

Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)