780 VOLUME 18 NUMBER 4 | apRiL 2010 | www.obesityjournal.org articles nature publishing group intervention and Prevention IntroductIon Obesity-induced inflammation plays a crucial role in the development of metabolic diseases such as insulin resistance, type 2 diabetes, hepatic steatosis, and cardiovascular dis- eases (1,2). Studies have shown that dysregulation of adipose tissue���derived proteins such as cytokines/chemokines and adipocytokines results in impaired insulin signaling and lipid metabolism (3,4). For example, adipose tissue���derived tumor necrosis factor-�� (TNF��) impairs insulin signaling by increas- ing serine phosphorylation of insulin receptor substrate-1, and thus induces insulin resistance (5���7). Mice rendered obese by a high-fat diet are protected from insulin resistance and type 2 diabetes by a defect in TNF�� and/or blockade of TNF�� activ- ity (8). Adipose tissue���derived monocyte chemoattractant protein-1 (MCP-1), which triggers adipose tissue inflammation by enhancing macrophage infiltration into adipose tissue (9), also impairs insulin signaling (10���12). On the other hand, adi- ponectin inhibits endogenous glucose production in the liver and reduces obesity-associated insulin resistance (13). Several lines of evidence indicate that TNF��, interleukin (IL)-6, and MCP-1 are upregulated in adipose tissue and plasma by obes- ity and in type 2 diabetes, whereas adiponectin is downregu- lated (1,5,12,14���16). Moreover, adipose tissue���derived factors including free fatty acids alter hepatic metabolism via their paracrine action, leading to abnormal fat accumulation and hepatic insulin resistance (17). It has been shown that TNF�� and IL-6 are key mediators of hepatic inflammation, liver cell death, fibrosis, and liver regeneration after injury (18), Dietary Capsaicin Reduces Obesity-induced insulin Resistance and Hepatic Steatosis in Obese Mice Fed a High-fat Diet Ji-Hye Kang1, Goto Tsuyoshi2,5, In-Seob Han3, Teruo Kawada2, Young Min Kim4 and Rina Yu1 Obesity-induced inflammation contributes to the development of obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, fatty liver disease, and cardiovascular disease. In this study, we investigated whether dietary capsaicin can reduce obesity-induced inflammation and metabolic disorders such as insulin resistance and hepatic steatosis. Male C57BL/6 obese mice fed a high-fat diet for 10 weeks received a supplement of 0.015% capsaicin for a further 10 weeks and were compared with unsupplemented controls. Glucose intolerance was estimated by glucose tolerance tests. Transcripts of adipocytokine genes and the corresponding proteins were measured by reverse transcription-PCR and enzyme-linked immunosorbent assay, and macrophage numbers were determined by flow cytometric analysis. Transient receptor potential vanilloid type-1 (TRPV-1), peroxisome proliferator���activated receptor (PPAR)-��, and PPAR�� coactivator-1�� (PGC-1��) mRNAs were also measured by RT-PCR, and PPAR�� luciferase assays were performed. Dietary capsaicin lowered fasting glucose, insulin, leptin levels, and markedly reduced the impairment of glucose tolerance in obese mice. Levels of tumor necrosis factor-�� (TNF��), monocyte chemoattractant protein-1 (MCP-1), and interleukin (IL)-6 mRNAs and proteins in adipose tissue and liver decreased markedly, as did macrophage infiltration, hepatic triglycerides, and TRPV-1 expression in adipose tissue. At the same time, the mRNA/protein of adiponectin in the adipose tissue and PPAR��/PGC-1�� mRNA in the liver increased. Moreover, luciferase assays revealed that capsaicin is capable of binding PPAR��. Our data suggest that dietary capsaicin may reduce obesity-induced glucose intolerance by not only suppressing inflammatory responses but also enhancing fatty acid oxidation in adipose tissue and/or liver, both of which are important peripheral tissues affecting insulin resistance. The effects of capsaicin in adipose tissue and liver are related to its dual action on PPAR�� and TRPV-1 expression/activation. Obesity (2010) 18, 780���787. doi:10.1038/oby.2009.301 1Department of Food Science and Nutrition, University of Ulsan, Ulsan, South Korea 2Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan 3Department of Biological Science, Ulsan, South Korea 4Department of Pathology, University of Ulsan College of Medicine, Ulsan, South Korea. 5Present address: Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan Correspondence: Rina Yu (rinayu@ulsan.ac.kr) Received 10 February 2009 accepted 24 July 2009 published online 1 October 2009. doi:10.1038/oby.2009.301

obesity | VOLUME 18 NUMBER 4 | apRiL 2010 781 articles intervention and Prevention indicating that metabolic dysregulation may be aggravated systemically by these adipose tissue���derived factors. In this regards, reducing obesity-induced inflammation by targeting the adipose tissue���derived proteins may be a useful strategy for preventing obesity-induced metabolic pathologies. Indeed, representative drugs such as thiazolinediones, which are widely used for treatment of insulin resistance and type 2 diabetes, elicit anti-inflammatory effects by depress- ing levels of inflammatory adipocytokines (e.g., TNF�� and IL-6) and by enhancing adiponectin levels (19). Recently curcumin, a naturally occurring anti-inflammatory phyto- chemical, has been shown to elicit hypoglycemic activity in type 2 diabetic KK-A(y) mice (20). Moreover, dietary cur- cumin reduces obesity-induced inflammation and diabetes in hereditary and diet-induced obese mice, and its action is associated with reversing adipocytokine dysregulation (21). These findings suggest that the antidiabetic activity of this compound is associated with its effects on inflammation and adipocytokine production. Capsaicin, a spicy component of hot pepper, is consumed extensively in several countries to enhance spicy flavor. In connection with the effect of cap- saicin on adipose tissue inflammation, our previous in vitro study showed that this agent inhibited the expression of inflammatory adipocytokines and release of the correspond- ing proteins (e.g., IL-6 and MCP-1) from obese adipose tis- sues and isolated adipocytes, while enhancing the expression and release of adiponectin (22). In addition, capsaicin inhib- ited macrophage responses, which are crucial for augment- ing adipose tissue inflammation (22). These in vitro findings strongly suggest that capsaicin should be useful in the treat- ment of obesity-related complications such as insulin resist- ance, which is triggered by obesity-induced inflammation. However, it remains unclear whether dietary supplementation with capsaicin can attenuate obesity-induced inflammation, and particularly whether it reduces obesity-related inflam- matory complications such as insulin resistance. To address these questions, we examined whether obesity-induced inflammatory responses are altered by dietary capsaicin. We fed mice with a high-fat diet containing 0.015% capsaicin, as consumed in the daily diet in various Asian countries includ- ing Korea (23), and examined obesity-induced inflammatory effects including insulin resistance. Our experiments revealed that dietary capsaicin reduced obesity-induced glucose intolerance and hepatic steatosis by modulating inflammatory responses and fatty acid oxidation in both adipose tissue and liver. Hence, capsaicin may be useful as a dietary additive for reducing obesity-induced metabolic disorders. Methods and Procedures animal experiments Eight-week-old male C57BL/6 mice were purchased from Hyochang (Daegu, Korea). All were maintained in specific pathogen-free condi- tions in the animal facility of the Immunomodulation Research Center (University of Ulsan, Ulsan, Korea). All experimental procedures were approved by the University of Ulsan Animal Care and Use Committee, and conformed to National Institutes of Health guidelines. Mice were fed a high-fat diet (45% calories from lard and soybean oil) (Research Diet, New Brunswick, NJ) 21% energy as fat 48% as carbohydrate 17% as protein, and 0.15% as cholesterol) for a total of 20 weeks. After 10 weeks of feeding, the high-fat diet���fed mice were divided into two groups: a control group (Obese, n = 6) and a group supplemented with 0.015% dietary capsaicin (obese+CAP, n = 6) (Wako, Osaka, Japan) for another 10 weeks. During the 10���20 weeks of capsaicin supplementa- tion, the obese control were daily given the same amounts of diet that were consumed by the capsaicin-supplemented obese mice for 24 h. Both sets of mice received water ad libitum. Thereafter, they were killed under CO2 anesthesia, and their adipose tissues and other organs were isolated, and samples were stored in RNAlater (Ambion, Austin, TX) for subsequent isolation of RNA and analysis of gene expression, or frozen in liquid nitrogen and stored at ���80 ��C, or in 10% buffered neutral formalin for histological evaluation. oral glucose tolerance test Oral glucose tolerance tests were performed on mice fasted for 16 h after 8 weeks of dietary capsaicin supplementation. Blood sam- ples were drawn from tail veins 0, 15, 30, 60, 90, and 120 min after administration of glucose (1 g/kg) to measure plasma glucose levels. The blood was collected in heparinized tubes and plasma was pre- pared immediately by centrifugation (3,000 r.p.m., 4 ��C, 15 min). Glucose levels were determined with a glucometer (Accu-Chek Roche Diagnostics, Indianapolis, IN) and/or by biochemical assay. Plasma insulin was measured by enzyme-linked immunosorbent assay. The assays were conducted with a mouse insulin kit (Crystal Chem, Downer���s Grove, IL). Insulin was quantified using a standard curve obtained with the SOFTmax curve-fitting program (Molecular Devices, Sunnyvale, CA). rt-Pcr analysis Total RNA was extracted from 50���100 mg tissue samples using a tri- reagent kit (MCR, Memphis, TN). Total RNA (0.5 ��g) was used for reverse transcription it was amplified by PCR in a single reaction (reverse transcription-PCR (RT-PCR)), using the Access RT-PCR sys- tem according to the manufacturer���s instructions in a TaKaRa Thermal cycler (TaKaRa Biomedicals, Tokyo, Japan). For semiquantitative analysis, the linearity of the amplification of MCP-1, IL-6, adiponec- tin, peroxisome proliferator���activated receptor-�� (PPAR��), PPAR�� coactivator-1�� (PGC-1��), ��-actin, and 36B4 complementary DNAs was established in preliminary experiments. The following sets of primers were used in the PCR amplifications. Amplification products obtained by PCR were electrophoretically separated on a 2% agarose gel. SYBR green-stained bands were photographed with a DS-34 Polaroid camera (Polaroid, Cambridge, MA). The intensities of the bands were measured with an NIH Image analyzer (Biocompare, San Francisco, CA). MCP-1, IL-6, adiponectin, PPAR��, and PGC-1�� signals were normalized to the mRNA level of the housekeeping gene 36B4, and expressed as ratios. Measurement of McP-1, IL-6, adiponectin, tnF�� proteins, and leptin Levels of MCP-1, IL-6, adiponectin, TNF��, and leptin were measured by enzyme-linked immunosorbent assay. The assays were conducted with an OptEIA mouse MCP-1 set, a mouse TNF�� set (BD Biosciences Pharmingen, San Diego, CA), a mouse IL-6 set (R&D Systems, Minneapolis, MN), a mouse adiponectin set (R&D Systems), and a mouse leptin kit (Crystal Chem). Samples were thawed, appropriately diluted with assay diluent, and assayed. MCP-1, IL-6, adiponectin, TNF��, and leptin were quantified using a standard curve obtained with the SOFTmax curve-fitting program (Molecular Devices). Flow cytometric analysis Stromal vascular cells isolated from adipose tissue were incubated in the dark at 4 ��C on a bidirectional shaker for 30 min in Fc-blocking solution (eBioscience, San Diego, CA), then stained with phycoerythrin- conjugated antimouse F4/80 (eBioscience) and/or fluorescein