Artemisia iwayomogi Extract Attenuates High-Fat Diet-Induced Obesity by Decreasing the Expression of Genes Associated with Adipogenesis in Mice

The objective of the present study was to determine whether Artemisia iwayomogi (AI) extract reduces visceral fat accumulation and obesity-related biomarkers in mice fed a high-fat diet (HFD), and if so, whether these effects are exerted by modulation of the expression of genes associated with adipogenesis and inflammation. AI extract supplementation for 11 weeks significantly prevented HFD-induced increments in body weight, visceral adiposity, adipocyte hypertrophy, and plasma levels of lipids and leptin. Additionally, AI extract supplementation resulted in downregulation of adipogenic transcription factors (PPARγ2 and C/EBPα) and their target genes (CD36, aP2, and FAS) in epididymal adipose tissue compared to the HFD alone. The AI extract effectively reversed the HFD-induced elevations in plasma glucose and insulin levels and the homeostasis model assessment of insulin resistance index. Furthermore, the extract significantly decreased gene expression of proinflammatory cytokines (TNFα, MCP1, IL-6, IFNα, and INFβ) in epididymal adipose tissue and reduced plasma levels of TNFα and MCP1 as compared to HFD alone. In conclusion, these results suggest that AI extract may prevent HFD-induced obesity and metabolic disorders, probably by downregulating the expression of genes related to adipogenesis and inflammation in visceral adipose tissue.


Introduction
Adipogenesis is the process by which mesenchymal precursor cells differentiate into adipocytes [1]. Although its presence is necessary in many ways, excess adipose tissue is associated with serious health problems such as obesity, cardiovascular disease, and type 2 diabetes [2,3]. Presently, CCAAT/enhancer binding protein (C/EBP ) and peroxisome proliferator-activated receptor 2 (PPAR 2) are considered the 2 primary transcription factors that mediate adipogenesis. It has been reported that inactivation of PPAR 2 and C/EBP in adipose tissue protects against obesity in rodent models [4][5][6]. us, potential therapeutic agents that have the ability to inhibit adipogenesis could have a profound impact as a strategy for preventing obesity and related metabolic disorders.
Adipose tissue not only serves as an organ for energy storage, but also as an endocrine organ by releasing various in�ammatory cytokines such as tumor necrosis factor (TNF ) and interleukin-(IL-)6 [7][8][9]. Proin�ammatory molecules produced by adipose tissue have been implicated as active participants in the development of in�ammation and the increased risk of obesity-related insulin resistance [9][10][11]. Increased production of monocyte chemoattractant protein 1 (MCP1), interferon (IFN) , IFN , TNF , and IL-6 in adipose tissue has been reported in animal models of obesity [8][9][10]12]. erefore, therapeutic agents that attenuate proin�ammatory cytokines may prove useful in the medical management of obesity-induced in�ammation.
Artemisia iwayomogi (AI), a member of Compositae, is a perennial herb easily found throughout Korea. It has been used as a traditional medicine and is known to have antiallergic, antiapoptotic, and antioxidant effects [13][14][15][16]. It has been reported that a carbohydrate fraction from AI suppresses spontaneous or 2,3,7,8-tetrachlorodibenzo-pdioxin-induced apoptotic death of mouse thymocytes [14,15]. Artemisia iwayomogi extract displays scavenging activity of peroxynitrite, a potent cytotoxic oxidant formed by the reaction between nitric oxide and superoxide radicals [16]. In addition, to our knowledge, few studies have described the bene�cial effects of AI in high-fat diet-(HFD-) fed obese rodents. One study reported that oral administration of AI extract signi�cantly reduced serum lipid levels in HFD-fed rats [17]. A recent study by Cho et al. reported that oral administration of AI extract to HFD-fed mice provoked upregulation of PPAR and its target genes involved in fatty acid oxidation in the skeletal muscle [18]. To date, however, no study has assessed the protective effects of AI extract on adipose tissue dysfunction in diet-induced obese animal models. erefore, the present study aimed to investigate whether AI extract could reduce visceral fat accumulation and improve obesity-related biomarkers in HFD-fed mice, and if so, whether these effects were exerted by modulation of the expression of genes associated with adipogenesis and in�ammation.

Preparation of AI Extract.
Artemisia iwayomogi was collected from Korean standard products in March 2008, and was identi�ed by Professor Wan Kyun Whang of the Pharmaceutical Botany Laboratory at Chung-Ang University in Seoul, Republic of Korea, where a voucher specimen has been deposited. Dried AI (500 g) was washed in water, oven dried at 40 ∘ C, mechanically fragmented, and then was powdered in an electric mill. e powder was extracted 5 times with ethanol (powder : solvent = 1 : 5) at room temperature. Aer �ltration, the extraction was vacuum concentrated to yield 6.25% ethanol extract (31.3 g), which was stored at −4 ∘ C until use.

HPLC Analysis.
Chromatography was performed using a Water HPLC system (Water Corporation, Milford, MA, USA) with an autosampler. HPLC separation was conducted using a Kromasil C18 column (4.6 mm × 250 mm, 5-m inner diameter) at 30 ∘ C with a �ow rate of 1.0 mL/min using a gradient mobile phase composed of water (A) and acetonitrile (B). e mobile phase comprised an 80 : 20 mixture of component A to B as the initial condition of the chromatographic run, and component B was increased to 80% in a linear gradient in 30 min. e sample injection volume was 10 L. e identities of the compounds in the AI extract were con�rmed by 1 H-NMR, 13 C-NMR, and MS, with individual purities of not less than 95%.

Animals and Experimental
Protocol. Male C57BL/6J mice (5 weeks old) were purchased from Orient Bio (Gyeonggi-do, Republic of Korea) and were maintained in 12 h light/dark with ad libitum access to food and water. Aer a 1-week acclimatization period, the mice were divided into 3 groups ( 1 per group): normal diet (ND), HFD, and 0.5% AI extract-supplemented diet (AED). e ND was a puri�ed diet based on the AIN-76 rodent diet composition. e HFD was identical to the ND, but to which 200 g fat/kg (170 g lard plus 30 g corn oil) and 1% cholesterol had been added. e AED was identical to the HFD but contained 0.5% (w/w) AI extract. e mice were fed the experimental diets for 11 weeks. Diet consumption was monitored daily, and body weight was monitored weekly. At the end of the feeding period, mice were anesthetized with diethyl ether aer an overnight fasting for 16 h, and their blood samples were collected in EDTA-coated tubes. Plasma samples were isolated by centrifugation at 4000 ×g for 20 min and stored at −80 ∘ C for subsequent analysis. Adipose and liver tissues were collected, washed with phosphatebuffered saline, and frozen at −80 ∘ C. Hepatic lipids were extracted as described by Folch et al. [19], using a chloroform-methanol mixture (2 : 1 v/v), and the dried lipid residues were dissolved in 2 mL ethanol. Concentrations of cholesterol, triglyceride, and free fatty acids in the hepatic lipid extracts were measured using the same enzymatic kits that were used for the plasma analysis.

Histological Analysis.
White adipose tissues (WATs) were �xed in neutral buffered formalin, embedded in para�n, and sectioned into 5 m sections onto slides. For histology, sections were stained with hematoxylin and eosin (H&E). e sectional areas of WAT were analyzed to quantify the size of the adipocytes.

RNA Extraction and Semiquantitative RT-PCR.
Total RNA was isolated from the epididymal adipose tissue of each mouse with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. Isolated RNA was quanti�ed using a spectrophotometer, and cDNA was synthesized using reverse transcriptase (Invitrogen). e PCR was programmed as follows: 10 min at 94 ∘ C; 30-33 cycles at 94 ∘ C for 30 s, 55 ∘ C for 30 s; 72 ∘ C for 1 min; 10 min incubation at 72 ∘ C. Four microliters of each PCR reaction were mixed with 1 L of 6-fold concentrated loading buffer and loaded onto 2% agarose gel containing ethidium bromide. e GenBank accession numbers of the relevant

Discussion
is feeding study was designed to assess whether AI extract supplementation for 11 weeks could improve diet-induced obesity and obesity-related biomarkers in mice. Based on a preliminary study involving different AI extract dosages (0.5%, 1%, and 2%), we determined that 0.5% AI extract was the minimal effective dose for preventing weight gain  . FER = (body weight gain for experimental period (g))/(food intake for experimental period (g)).
in HFD-fed mice (data not shown). Hence, the 0.5% AI extract was considered for this study. In the present study, the AI extract signi�cantly decreased not only body weight gain, but also visceral adiposity and adipocyte hypertrophy in HFD-fed mice. Since visceral adiposity is positively correlated to plasma leptin concentration, the circulating leptin level is an ideal indicator for assessing obesity in both experimental animals and humans [20,21]. In this context,  the lower plasma leptin level recorded in the AED-fed mice may be attributable to the prevention of visceral adipocyte hypertrophy.
As there was no signi�cant difference in food consumption between HFD-and AED-fed mice, the bene�cial effects of the AI extract on body weight gain and visceral fat accumulation evidently did not depend on decreased energy intake. us, we hypothesized that the AI extract reduced HFD-induced body weight gain and visceral fat accumulation by mediating the inhibition of adipogenesis. at the expression of PPAR 2 and C/EBP in WAT is upregulated in HFD-induced obese animals is well known [4,22]. Increased PPAR 2 and C/EBP function cooperatively to transactivate adipocyte genes, including FAS, CD36, and aP2 [23][24][25][26]. FAS catalyzes the reactions for the synthesis of long-chain fatty acids [27], whereas CD36 and aP2 facilitate the uptake of long-chain fatty acids in adipocytes [23,28], thereby increasing adipocyte size and fat accumulation. In the current study, the AI extract signi�cantly reversed the HFDinduced upregulation of adipogenic transcription factors (PPAR 2 and C/EBP ) and their target genes (FAS, CD36, and aP2) in the epididymal adipose tissue of mice. ese decreased expressions of adipogenic genes by the AI extract may have contributed to the lower visceral adiposity and body weight gain. In addition, the AI extract signi�cantly decreased plasma FFA levels in HFD-fed mice. As increased plasma concentrations of FFA form a vicious relationship with PPAR 2 activation in diet-induced obese animals [29,30], the decreased FFA level in the AED-fed mice might also be considered associated with PPAR 2 inactivation.
Increased adipose tissue accumulation in obese individuals correlates with the overproduction of proin�ammatory cytokines that play crucial roles in the development of obesity-induced in�ammation [8,11]. In obese animals, excess adipose tissue increases the expression and secretion of TNF , a prototypical in�ammatory cytokine [4,9,12]. In turn, increased TNF activates adipocytes, thereby further enhancing the expression of various proin�ammatory genes such as MCP1, IL-6, IFN , and IFN [31,32]. IL-6 induces a hepatic acute-phase reaction with upregulated acute-phase proteins, including C-reactive protein and �brinogen [33,34], whereas MCP1 contributes to macrophage in�ltration into adipose tissue, which leads to chronic in�ammation [35]. IFN / directly stimulates IFN production, a regulator of innate immune response, in T cells [36]. Our results suggested that the decrease in visceral adiposity by the AI extract might have contributed, in part, to the decreased expression of proin�ammatory cytokines (TNF , MCP1, IL-6, IFN , and IFN ) and reduced plasma levels of TNF and MCP1. Consequently, the decreased gene expression and secretion of proin�ammatory cytokines in the AED-fed mice may have contributed to the prevention of obesity-induced in�ammation.
Several studies have demonstrated that cytokines play crucial roles in the development of insulin resistance [35,[37][38][39]. In diet-induced obese animals, increased plasma levels of TNF , IL-6, MCP1, and leptin have been shown to impair the ability of insulin to activate signal transduction and stimulate glucose uptake into skeletal muscle and adipose tissue [35,[37][38][39][40][41]. us, in the present study, improvement of insulin resistance by the AI extract might be associated with decreased gene expression and/or production of cytokines such as TNF , IL-6, MCP1, and leptin.
Separation and determination of chemical constituents are generally recommended for standardization and quality control of herbal products and herb-related investigations [42]. Several studies have demonstrated that AI contains �avonoids such as genkwanin and �aceosidin [43], essential oils, including eugenol and 1,8-cineole [44], and coumarin compounds such as scopoletin and scopolin [43,45]. In addition, scopoletin has been used as a standard compound for the veri�cation and identi�cation of AI [46,47]. In the present study, we isolated not only scopoletin, but also scopolin and acetophenone glycoside from the AI extract ( Figure 1 and Table 2). Of these, scopolin (1.21%) was the most abundant, followed by scopoletin (0.38%) and acetophenone glycoside (0.26%). In addition, the scopolin content of the AI extract was higher in our study than that  [43,45]. is difference may be due to the differing extraction and isolation methods as well as differences in plant material [43,45]. Previous studies have shown that coumarin compounds from Fraxinus rhynchophylla [48], Angelica gigas [49], and Ionidium suffruticosum [50] inhibit adipocyte differentiation in 3T3-L1 cells and/or reduce body weight gain and plasma lipid levels in mice fed a high-fat diet. us, the antiobesity activity of AI extract observed in the present study may be attributable to the presence of high amounts of coumarin compounds in the plant. Further studies are needed to determine the major active compounds in AI extract that are responsible for decreased visceral adiposity and other obesity-related biomarkers.
In summary, the present study showed that AI extract reduced visceral fat accumulation in HFD-fed mice and improved the risk factors related to the metabolic syndrome, such as in�ammation and insulin resistance. e evidence obtained in this study suggests that the bene�cial effects of AI extract may be due to, at least in part, downregulation of the genes related to adipogenesis and in�ammation in the visceral adipose tissue of mice. erefore, dietary supplementation with this extract, if validated in human studies, may provide an adjunctive therapy for the prevention and/or treatment of obesity and metabolic syndrome.