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Naringenin chalcone improves adipocyte functions by enhancing adiponectin production
Molecular and Cellular Endocrinology 323 (2010) 208–214
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Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Naringenin chalcone improves adipocyte functions by enhancing adiponectin production Taro Horiba a,∗ , Ikuko Nishimura a , Yuji Nakai b , Keiko Abe b , Ryuichiro Sato b a b
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
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Article history: Received 3 December 2009 Received in revised form 20 February 2010 Accepted 25 March 2010 Keywords: Naringenin chalcone Adiponectin DNA microarray Adipocyte
a b s t r a c t Naringenin chalcone is a flavonoid contained in tomato peel. In this study, we investigated its effects on adipocyte functions related to metabolic processes, including adipocytokine production. Naringenin chalcone promoted the gene expression (8.0-fold, p < 0.001) and protein secretion (2.2-fold, p < 0.001) of adiponectin from 3T3-L1 adipocytes. Reporter gene assays revealed that naringenin enhanced the activity of peroxisome proliferator-activated receptor ␥. DNA microarray experiments and Gene Ontology analysis revealed that naringenin chalcone also up-regulated the genes associated with mitochondrial energy metabolism, reflecting its insulin-sensitizing effects. Conversely, genes in categories such as those for cell adhesion were down-regulated. The expression of one adiponectin receptor, AdipoR2, was also increased (1.8-fold, p < 0.01), suggesting that naringenin chalcone could activate the adiponectin pathway through the elevation of both the ligand and its receptor. These results indicate that naringenin chalcone is a potent tomato flavonoid that improves adipocyte metabolic functions and exerts insulin-sensitizing effects by activating an adiponectin-related pathway. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Obesity or excess accumulation of visceral fat is a risk factor for atherosclerosis, diabetes, hyperlipidemia, and metabolic syndrome (Matsuzawa, 2006). A wide variety of studies performed over more than a decade have shown that adipocytes not only store the excess energy as fat, but also function as an endocrine organ that secretes various bioactive molecules, known as adipocytokines (Shimomura et al., 1996). Adipocytokines play pivotal roles in the regulation of food intake, insulin sensitivity, and energy metabolism (Fasshauer and Paschke, 2003). Under obese conditions, adipose tissue is characterized by the enhanced infiltration of macrophages (Weisberg et al., 2003), and increased expression of tumor necrosis factor-␣ (TNF-␣) and monocyte chemoattractant protein-1 (MCP-1), which are implicated in insulin resistance and metabolic disorders (Hotamisligil et al., 1993; Sartipy and Loskutoff, 2003). Conversely, the gene expression and secretion of adiponectin, which is believed to be a phys-
Abbreviations: ANOVA, analysis of variance; ATP, adenosine 5 –triphosphate; DEGs, differentially expressed genes; DMEM, Dulbecco’s Modified Eagle Medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FDR, false discovery rate; GO, Gene Ontology; MCP-1, monocyte chemoattractant protein-1; PPAR␥, peroxisome proliferator-activated receptor ␥; PPRE, PPAR-responsive element; TNF-␣, tumor necrosis factor-␣. ∗ Corresponding author. Tel.: +81 4 7123 5961; fax: +81 4 7123 5961. E-mail address: [email protected] (T. Horiba). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.03.020
iologically active adipocytokine that not only improves insulin sensitivity but also inhibits the inflammatory process (Yamauchi et al., 2001; Diez and Iglesias, 2003), are decreased in obesity. This suggests that the modulation of adipocytokine secretion from adipose tissue is critical for preventing obesity-induced metabolic abnormalities. Tomato is a common and widely consumed vegetable containing various compounds with health benefits. It has been reported that regular tomato consumption decreases the incidence of cardiovascular diseases (Pandey et al., 1995) and platelet aggregation in type 2 diabetes (Lazarus et al., 2004). Naringenin chalcone is one of the predominant flavonoids found in tomatoes, and accumulates almost exclusively in the tomato peel. This flavonoid is an intermediate in the biosynthetic pathway for flavonols and is converted to naringenin by chalcone isomerase (Muir et al., 2001). We previously reported that naringenin chalcone suppressed allergic reactions by inhibiting histamine release (Yamamoto et al., 2004). It has also been reported that naringenin chalcone inhibits the production and secretion of TNF- ␣ and MCP-1 from macrophages (Hirai et al., 2007), and recent reports have shown that some flavonoids can modulate the secretion of adipocytokines from adipocytes (Hassan et al., 2007; Kunimasa et al., 2009). However, little is known about the regulation of gene expression in adipocytes by naringenin chalcone. The present study therefore aimed to clarify the effects of naringenin chalcone on adipocyte functions related to metabolic processes, including adipocytokine production. We examined the effects of naringenin chalcone on
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adiponectin production and the changes in patterns of gene expression using DNA microarray analysis of 3T3-L1 adipocytes. 2. Materials and methods
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to the manufacturer’s instructions. Twenty-four hours after transfection, the transfected cells were cultured in medium containing the indicated concentrations of naringenin chalcone for an additional 24 h. Luciferase reporter activities were quantified using the dual luciferase system (Promega, WI, USA), in accordance with the manufacturer’s protocols.
2.1. Materials 2.7. DNA microarray analysis Naringenin chalcone was prepared from tomato peel (Yamamoto et al., 2004). Briefly, tomato extracts containing polyphenols were extracted from tomato peel with 60% (v/v) ethanol at 60 ◦ C for 2 h. Subsequently, naringenin chalcone was purified from the crude extracts by high performance liquid chromatography fractionation, and was dissolved in dimethyl sulfoxide to prepare stock solutions (200 mM). All the other chemicals used were from Sigma (MO, USA). 2.2. Cell culture and treatment NIH-3T3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen Japan, Tokyo, Japan). 3T3-L1 preadipocytes were cultured in DMEM containing 10% bovine serum (Invitrogen Japan). Two days after confluence, cells were differentiated into adipocytes by the addition of differentiation medium (DMEM containing 10% FBS, 0.5 mM 3-isobuthyl1-methylxantine, 1 M dexamethasone, and 10 g/ml insulin) in the presence or absence of the indicated concentration of naringenin chalcone (day 0). After 2 days, the 3T3-L1 cells were transferred to adipocyte-growing medium (DMEM containing 10% FBS and 5 g/ml insulin) in the presence or absence of the indicated concentrations of naringenin chalcone (25–100 M), which was replenished every 2 days. Dimethyl sulfoxide was used as a vehicle control for naringenin chalcone (0.1%). Culture medium was collected on day 8 and the amount of adiponectin secreted during the last 48 h of differentiation (days 6–8) was measured by enzyme-linked immunosorbent assay (ELISA). Total RNA was extracted for quantitative reverse transcription-polymerase chain reaction (RT-PCR) and DNA microarray analysis. These experiments were performed in triplicate. 2.3. Measurement of adiponectin production The concentration of adiponectin in the culture medium was determined by ELISA. ELISAs were conducted using adiponectin ELISA kits (Otsuka, Tokyo, Japan). 2.4. RNA extraction Total RNA was isolated from untreated control and treated cells using TRIzol (Invitrogen Japan) and purified using an RNeasy mini Kit (Qiagen, Hilden, Germany). Only samples with a quality ratio of 1.6–2.0 measured at A260 nm/A280 nm were used for quantitative RT-PCR and DNA microarray experiments. 2.5. Quantitative RT-PCR Changes in the expression of the adiponectin and AdipoR2 gene were assessed using a SYBR® PrimeScript® RT-PCR Kit (Takara-Bio, Shiga, Japan), according to the manufacturer’s instructions. Briefly, total RNA was reverse-transcribed into complementary DNA using a PrimeScript® RT reagent Kit (Takara-Bio). Subsequent real-time quantitative RT-PCR was performed with SYBR® Premix Ex TaqTM (TakaraBio), with PCR amplification carried out for 1 cycle at 95 ◦ C for 10 s, then 45 cycles at 95 ◦ C for 5 s and at 60 ◦ C for 20 s. Subsequently, a dissociation curve was generated to ensure amplification of only the proper PCR product. All real-time reactions were carried out on an MxPro 3000P QPCR System (Stratagene, CA, USA), and analysis was done using MxPro software (Stratagene). Relative expression levels were calculated according to standard curves obtained from real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. The primer sequences used were: adiponectin 5 -GAT GGC AGA GAT GGC ACT CC-3 (forward) and 5 -CTT GCC AGT GCT GCC GTC AT-3 (reverse), AdipoR2 5 -CTG TAA CCC ACA ACC TTG CT -3 (forward) and 5 -GGA ACC CTT CTG AGA TGA CA-3 (reverse), cyclophilin 5 -TGG TGA CTT TAC ACG CCA TA-3 (forward) and 5 -CAG TCT TGG CAG TGC AGA TA-3 (reverse). 2.6. Luciferase reporter assay A luciferase reporter assay was performed as described previously (Arimura et al., 2004; Kanayama et al., 2007). Briefly, in assays to measure the transcription of the adiponectin and perilipin genes, pGL4-adiponectin (a luciferase reporter plasmid containing about 1000 bp of the adiponectin 5 -flanking region) or pGL3perilipin (a luciferase reporter plasmid containing the segment around −2000 bp of the perilipin 5 -flanking region), pCMX-PPAR␥ (an expression plasmid for peroxisome proliferator-activated receptor ␥, PPAR␥), and pRL-CMV (an internal control plasmid) were transfected into NIH-3T3 cells cultured on 12-well plates. In assays using the GAL4-PPAR␥ chimeric protein, pG5-Luc (a reporter plasmid containing consensus Gal4-binding sites), pM-PPAR␥ (an expression plasmid for a chimeric protein for a GAL4 DNA-binding domain and PPAR␥ ligand-binding domain), and pRL-CMV were transfected into NIH-3T3 cells cultured on 12-well plates. Transfections were performed using Lipofectamine 2000 (Invitrogen Japan), according
DNA microarray analysis was performed using Mouse Genome 430 2.0 Array (Affymetrix Santa Clara, CA, USA), according to the manufacturer’s protocol. Briefly, 2 g of total RNA was reverse-transcribed into cDNA using a poly(dT) oligonucleotide attached to a T7 promoter and converted to dsDNA, which was then used as a template for the synthesis of biotinylated cRNA by T7 RNA polymerase. Labeled cRNA was fragmented and hybridized to the array at 45 ◦ C for 16 h. After hybridization, the array was washed and stained with streptavidin-phycoerythrin. Fluorescent signals were scanned using the Affymetrix GeneChip System. 2.8. Analysis of DNA microarray data Affymetrix GCOS software was used to reduce the array images to the intensity of each probe (CEL files). The CEL files were quantified using the DFW algorithm (Chen et al., 2007) using the statistical language R (Ihaka and Gentleman, 1996) and Bioconductor (Gentleman et al., 2004). The differentially expressed genes were identified using Rank Products (Breitling et al., 2004). The annotation file for the Mouse Genome 430 2.0 Array was downloaded from the Affymetrix website. DAVID was used to detect over-represented Gene Ontology (GO) categories in each group of differentially expressed genes (DEGs) (Dennis et al., 2003; Huang da et al., 2009). We compared the DEGs with the Mouse Genome 430 2.0 Array background. The functional annotation chart, a tool integrated in DAVID, was applied to examine significantly over-represented GO categories. The Benjamini and Hochberg FDR correction (Benjamini and Hochberg, 1995) was employed to correct for multiple testing. An FDR-corrected p value < 0.01 was accepted as significant. 2.9. Statistical analysis All values were represented as means ± standard deviations. Unpaired Student’s t-tests were used to analyze differences between control and treated cells. One-way analysis of variance (ANOVA) was used for experiments with three or more groups, followed by Fisher’s protected least significant difference test, with p < 0.05 accepted as significant.
3. Results 3.1. Naringenin chalcone enhances the genetic expression of adiponectin and promotes adiponectin secretion from 3T3-L1 adipocytes We first examined the effects of naringenin chalcone on the expression and production of adiponectin in cultured 3T3L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes in the presence or absence of the indicated concentrations of naringenin chalcone for 8 days. Naringenin chalcone significantly increased adiponectin mRNA levels up to 8.0-fold in a dose-dependent manner (p < 0.001) (Fig. 1A). The results of ELISAs showed that the amount of adiponectin secreted during the last 48 h of differentiation (days 6–8) was also increased (up to 2.2-fold, p < 0.001) by naringenin chalcone treatment (Fig. 1B). 3.2. Naringenin chalcone stimulates the activity of PPAR We carried out luciferase assays using a reporter gene including the adiponectin 5 -flanking region, which contains the PPARresponsive element (PPRE), to examine if naringenin chalcone stimulated the transcription of the adiponectin gene. NIH-3T3 cells were co-transfected with the adiponectin reporter plasmid and the expression plasmid for PPAR␥, which plays critical roles in the transcriptional activation of adipocytokines including adiponectin (Lehmann et al., 1995; Combs et al., 2002). The cells were cultured with naringenin chalcone for the last 24 h. Naringenin chalcone dose-dependently enhanced luciferase activity by up to 1.5-fold (p < 0.01) (Fig. 2A). The same results were obtained using the perilipin promoter reporter plasmid (up to 1.4-fold, p < 0.01), which also contains a functional PPRE (Arimura et al., 2004) (Fig. 2B).
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Fig. 1. Naringenin chalcone induces adiponectin expression. 3T3-L1 cells were treated with naringenin chalcone at the indicated concentration throughout a period of differentiation (8 days). (A) Total RNA was prepared 8 days after differentiation induction. Relative mRNA levels of adiponectin were calculated according to standard curves obtained by real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. (B) Adiponectin secretion levels during the last 48 h of differentiation. Six days after differentiation induction, 3T3-L1 adipocytes were incubated in medium containing 10% FBS and the indicated dose of naringenin chalcone for 48 h, and the medium was then collected to measure the amount of secreted adiponectin. The control value was taken as 100%. The data are shown as the means ± S.D. of triplicate cultures.
Fig. 2. Naringenin chalcone stimulates PPAR␥-mediated transcription. NIH-3T3 cells were transfected with one of the reporter plasmids for adiponectin (A) and perilipin (B) together with pRL-CMV (an internal control plasmid) and expression plasmid for PPAR␥. (C) NIH-3T3 cells were transfected with pG5-Luc (a reporter plasmid containing consensus Gal4-binding sites), pM-PPAR␥ (an expression plasmid for a fusion protein, the Gal4 DNA-binding domain and PPAR␥ ligand-binding domain), and pRL-CMV. Twenty-four hours after transfection, cells were treated with naringenin chalcone at the indicated concentration for 24 h. Luciferase reporter activities were then quantified. The control value was taken as unity. The data are shown as the means ± S.D. of triplicate cultures.
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Table 1 Significantly enriched GO terms (metabolic process and its child terms) found in top 1153 up-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
These data indicate that naringenin chalcone augmented the genetic expression of PPAR␥ targets. To confirm the direct effect of naringenin chalcone on the transcriptional activity of PPAR␥, we employed a heterologous Gal4 system. In this assay, luciferase gene transcription is driven by a promoter containing consensus Gal4-binding sites, with no co-regulatory transcription factors. NIH-3T3 cells were transfected with a reporter plasmid (pG5-Luc) and the expression plasmid encoding the PPAR␥ ligand-binding domain coupled to the DNA-binding domain of Gal4 (pM-PPAR␥). Naringenin chalcone significantly enhanced the transcription of the reporter gene in a dose-dependent manner (up to 1.8-fold, p < 0.001) (Fig. 2C). These results indicate that naringenin chalcone accelerated the transcription of adiponectin by stimulating the transcriptional activity of PPAR␥. 3.3. DNA microarray analysis and GO on up- or down-regulated genes We performed DNA microarray experiments to explore the effects of naringenin chalcone on adipocyte functions. By applying R statistical software (Ihaka and Gentleman, 1996), we found that 1153 transcripts were up-regulated and 1031 transcripts were down-regulated in naringenin chalcone-treated adipocytes, compared with control, untreated adipocytes (FDR < 0.05). Using the DAVID bioinformatics resources (Dennis et al., 2003; Huang da et al., 2009), we compared the gene lists mentioned above with the Mouse Genome 430 2.0 Array background. To extract statistically over-represented categories from the gene lists, we applied the DAVID functional annotation chart tool. The relationships between significantly enriched categories are shown in supplemental Fig.
S1. In the hierarchical structure of GO, the more specific categories appear in the deeper hierarchy. Categories with more important biological meanings therefore appear at the lower end of the graph. The significantly enriched categories of genes that were up-regulated by naringenin chalcone treatment are summarized in Tables 1 and 2. Genes related to mitochondrial energy production (‘ubiquinone biosynthetic process’, ‘mitochondrial electron transport, NADH to ubiquinone’, ‘ATP synthesis coupled proton transport’, ‘mitochondrial membrane organization’) were significantly up-regulated in the naringenin chalcone-treated adipocytes (p = 2.0E-03, 7.3E-05, 5.0E-03, and 1.1E-03, respectively). There was also significant up-regulation of genes associated with ‘glucose catabolic process’, ‘fatty acid beta-oxidation’ and ‘tricarboxylic acid cycle’ (p = 2.0E-03, 3.7E-03, and 4.4E-14, respectively). These observations suggest that naringenin chalcone enhances energy production by stimulating glucose utilization, fatty acid oxidation, and oxidative phosphorylation in adipocytes. Gene categories related to ‘cell adhesion’, ‘development’, and ‘morphogenesis’ were conversely down-regulated by naringenin chalcone treatment (Table 3). The results of this microarray experiment also confirmed the significant up-regulation of adiponectin transcripts in naringenin chalcone-treated adipocytes. Naringenin chalcone treatment also stimulated the genetic expression of one of the adiponectin receptors, AdipoR2. The results of quantitative RT-PCR analysis confirmed significant up-regulation of AdipoR2 expression (1.8-fold, p < 0.01) (Fig. 3). These results suggest that naringenin chalcone is capable of enhancing adiponectin functions in adipocytes via both increased adiponectin secretion and increased receptor expression.
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T. Horiba et al. / Molecular and Cellular Endocrinology 323 (2010) 208–214 Table 2 Significantly enriched GO terms (cellular process and its child terms) found in top 1153 up-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
Table 3 Significantly enriched GO terms found in top 1031 down-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
4. Discussion
Fig. 3. Naringenin chalcone enhances AdipoR2 expression. 3T3-L1 cells were treated with or without 100 M naringenin chalcone throughout a period of differentiation (8 days). Total RNA was prepared 8 days after differentiation induction. Relative mRNA levels of AdipoR2 were calculated according to standard curves obtained by real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. The control value was taken as 100%. The data are shown as the means ± S.D. of triplicate cultures.
The current study found that naringenin chalcone enhanced adiponectin gene expression and its secretion from 3T3-L1 adipocytes. It is well established that adiponectin gene expression is regulated by PPAR␥, and that a functional PPRE is present in the 5 -flanking promoter region of the adiponectin gene (Iwaki et al., 2003). PPAR␥ has been shown to be one of key regulators of adipocyte differentiation, and compounds such as thiazolidinediones stimulate PPAR␥ transcriptional activity and improve adipocyte functions by increasing the expression levels of genes associated with lipid metabolism (Ferre, 2004). The results of luciferase reporter gene assays in this study confirmed that naringenin chalcone enhanced PPAR␥-mediated transcription of both the adiponectin and perilipin genes. Consistent with enhanced PPAR␥ transcriptional activity, naringenin chalcone promoted differentiation of adipocytes, assessed by triglyceride accumulation, with no notable toxicity at concentrations up to 100 M (data not shown). It is likely that the increase in adiponectin gene expression in 3T3-L1 adipocytes was responsible for the elevated secretion of adiponectin into the culture medium. Plasma adiponectin levels were increased when naringenin chalcone was orally administered
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to diabetic KK-Ay mice in our preliminary experiments (data not shown). In addition, naringenin chalcone has been reported to have inhibitory effects on the secretion of TNF␣- and MCP-1, which are associated with induction of insulin resistance (Hirai et al., 2007). Overall, these results suggest that naringenin chalcone is capable of enhancing insulin sensitivity via modulating the expression and secretion of various adipocytokines in adipose tissues. DNA microarray analysis revealed that various clusters of genes related to energy metabolism were simultaneously up-regulated, whilst a few categories related to functions such as cell adhesion and developmental processes were down-regulated by naringenin chalcone treatment. It has previously been reported that the expression of genes related to cell adhesion were down-regulated by the activation of PPAR␥ (Galli et al., 2002). Thus, it is possible that naringenin chalcone could exert certain physiological effects due to the reduced expression of these genes, in addition to its effects due to increased adiponectin gene expression. Interestingly, the expression of genes involved in oxidative phosphorylation was markedly augmented in naringenin chalconetreated adipocytes. The oxidative phosphorylation pathway is the process by which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers. This process takes place in mitochondria and is the major source of the ATP supply in aerobic organisms. Previous studies have reported that mitochondrial activity is impaired (Morino et al., 2005; Kelley et al., 2002; Petersen et al., 2005) and that the mRNA levels of genes associated with mitochondrial oxidative metabolism are reduced in skeletal muscle of insulin-resistant subjects (Mootha et al., 2003; Patti et al., 2003). Another report demonstrated a significant reduction in the expression of genes encoding components of mitochondrial oxidative phosphorylation in adipose tissue from obese monozygotic twins (Mustelin et al., 2008). These results indicate that mitochondrial dysfunction and defects in expression of genes involved in oxidative phosphorylation are characteristic of insulin resistance. Our current results suggest the possibility that naringenin chalcone is capable of enhancing insulin sensitivity by improving mitochondrial energy metabolism in adipocytes. Two types of cell surface receptors for adiponectin, AdipoR1 and AdipoR2, have so far been identified (Yamauchi et al., 2003). AdipoR1 has been identified primarily in skeletal muscles, while AdipoR2 is most abundant in the liver in mice. Expression of AdipoR receptors in mouse adipose tissue has also been reported, with a decreased expression of these receptors in insulin-resistant mice (Tsuchida et al., 2004). The results of our microarray and quantitative RT-PCR experiments indicated that the genetic expression of AdipoR2 was enhanced in naringenin chalcone-treated adipocytes, suggesting that naringenin chalcone affects adipocyte functions by stimulating the autocrine and/or paracrine effects of adiponectin. This model could account for the observed increase in expression of genes involved in fatty acid beta-oxidation in naringenin chalcone-treated adipocytes. Since the genetic expression of AdipoR in adipose tissue is not regulated by thiazolidinediones (Li et al., 2007), the increased expression of AdipoR2 by naringenin chalcone is unlikely to be mediated through PPAR␥ activation. The molecular mechanisms responsible for the enhancement of AdipoR2 gene expression by naringenin chalcone is now under investigation. Recent studies have uncovered relationships between caloric restriction, adipocytokine regulation, and the regulation of lipid metabolism. Caloric restriction increases plasma adiponectin and improves lipid metabolism in growth hormone transgenic mice (Wang et al., 2007). Another study demonstrated that caloric restriction significantly increased adipose tissue contents of adiponectin, thereby stimulating the expression of genes involved in fatty acid oxidation and ameliorating insulin resistance (Zhu et al., 2007). Many of the changes in gene expression mentioned above occurred in naringenin chalcone-treated adipocytes in our
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study. Although our study was limited by the use of an adipocyte cell line, rather than adipose tissues from animals fed naringenin chalcone, the results suggest that naringenin chalcone exerts beneficial effects on adipocyte functions by mimicking the effects of caloric restriction in adipose tissue. Naringenin chalcone has been detected as a glucuronide conjugate in rat plasma (Yoshimura et al., 2009), while another study demonstrated that flavonoid metabolites could be deconjugated during inflammation, and contribute to its aglycone bioactivity (Kawai et al., 2008). Overall, these results suggest that orally administered naringenin chalcone is capable of altering gene expression in adipose tissue as follows: (1) Naringenin chalcone is absorbed and metabolized into glucuronide conjugate; (2) it is delivered to the target sites, such as white adipose tissues; (3) it is converted to aglycone, and regulates gene expression. However, fasting has been shown to augment hepatic expression of AdipoR2 in young pigs (Liu et al., 2008). Considering the up-regulation of AdipoR2 in adipocytes by naringenin chalcone and its putative mimicking of the effects of caloric restriction, it is possible that naringenin chalcone could also affect hepatic energy metabolism. Further studies are required to elucidate the effects of naringenin chalcone on whole-body energy metabolism. In conclusion, the tomato flavonoid naringenin chalcone stimulates adiponectin gene expression in adipocytes through PPAR␥ activation. Naringenin chalcone also enhances the genetic expression of AdipoR2 in adipocytes, thereby amplifying its effects on adiponectin. The results of DNA microarray analysis indicate that transcriptional alterations in mitochondrial oxidative metabolism caused by naringenin chalcone may produce desirable insulinsensitizing effects. These results suggest that eating naringenin chalcone could contribute to the prevention and improvement of insulin resistance and related metabolic syndrome. Acknowledgments We thank Dr. A. Obata and Dr. A. Matsuyama for valuable suggestions and discussions, and Mr. T. Manaka and Mr. K. Okada for naringenin chalcone preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2010.03.020. References Arimura, N., Horiba, T., Imagawa, M., Shimizu, M., Sato, R., 2004. The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes. J. Biol. Chem. 279, 10070–10076. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300. Breitling, R., Armengaud, P., Amtmann, A., Herzyk, P., 2004. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett. 573, 83–92. Chen, Z., McGee, M., Liu, Q., Scheuermann, R.H., 2007. A distribution free summarization method for Affymetrix GeneChip arrays. Bioinformatics 23, 321–327. Combs, T.P., Wagner, J.A., Berger, J., Doebber, T., Wang, W.J., Zhang, B.B., Tanen, M., Berg, A.H., O’Rahilly, S., Savage, D.B., Chatterjee, K., Weiss, S., Larson, P.J., Gottesdiener, K.M., Gertz, B.J., Charron, M.J., Scherer, P.E., Moller, D.E., 2002. Induction of adipocyte complement-related protein of 30 kilodaltons by PPARgamma agonists: a potential mechanism of insulin sensitization. Endocrinology 143, 998–1007. Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A., 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, P3. Diez, J.J., Iglesias, P., 2003. The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur. J. Endocrinol. 148, 293–300. Fasshauer, M., Paschke, R., 2003. Regulation of adipocytokines and insulin resistance. Diabetologia 46, 1594–1603. Ferre, P., 2004. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes 53 (Suppl. 1), S43–S50.
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Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Naringenin chalcone improves adipocyte functions by enhancing adiponectin production Taro Horiba a,∗ , Ikuko Nishimura a , Yuji Nakai b , Keiko Abe b , Ryuichiro Sato b a b
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
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Article history: Received 3 December 2009 Received in revised form 20 February 2010 Accepted 25 March 2010 Keywords: Naringenin chalcone Adiponectin DNA microarray Adipocyte
a b s t r a c t Naringenin chalcone is a flavonoid contained in tomato peel. In this study, we investigated its effects on adipocyte functions related to metabolic processes, including adipocytokine production. Naringenin chalcone promoted the gene expression (8.0-fold, p < 0.001) and protein secretion (2.2-fold, p < 0.001) of adiponectin from 3T3-L1 adipocytes. Reporter gene assays revealed that naringenin enhanced the activity of peroxisome proliferator-activated receptor ␥. DNA microarray experiments and Gene Ontology analysis revealed that naringenin chalcone also up-regulated the genes associated with mitochondrial energy metabolism, reflecting its insulin-sensitizing effects. Conversely, genes in categories such as those for cell adhesion were down-regulated. The expression of one adiponectin receptor, AdipoR2, was also increased (1.8-fold, p < 0.01), suggesting that naringenin chalcone could activate the adiponectin pathway through the elevation of both the ligand and its receptor. These results indicate that naringenin chalcone is a potent tomato flavonoid that improves adipocyte metabolic functions and exerts insulin-sensitizing effects by activating an adiponectin-related pathway. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Obesity or excess accumulation of visceral fat is a risk factor for atherosclerosis, diabetes, hyperlipidemia, and metabolic syndrome (Matsuzawa, 2006). A wide variety of studies performed over more than a decade have shown that adipocytes not only store the excess energy as fat, but also function as an endocrine organ that secretes various bioactive molecules, known as adipocytokines (Shimomura et al., 1996). Adipocytokines play pivotal roles in the regulation of food intake, insulin sensitivity, and energy metabolism (Fasshauer and Paschke, 2003). Under obese conditions, adipose tissue is characterized by the enhanced infiltration of macrophages (Weisberg et al., 2003), and increased expression of tumor necrosis factor-␣ (TNF-␣) and monocyte chemoattractant protein-1 (MCP-1), which are implicated in insulin resistance and metabolic disorders (Hotamisligil et al., 1993; Sartipy and Loskutoff, 2003). Conversely, the gene expression and secretion of adiponectin, which is believed to be a phys-
Abbreviations: ANOVA, analysis of variance; ATP, adenosine 5 –triphosphate; DEGs, differentially expressed genes; DMEM, Dulbecco’s Modified Eagle Medium; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FDR, false discovery rate; GO, Gene Ontology; MCP-1, monocyte chemoattractant protein-1; PPAR␥, peroxisome proliferator-activated receptor ␥; PPRE, PPAR-responsive element; TNF-␣, tumor necrosis factor-␣. ∗ Corresponding author. Tel.: +81 4 7123 5961; fax: +81 4 7123 5961. E-mail address: [email protected] (T. Horiba). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.03.020
iologically active adipocytokine that not only improves insulin sensitivity but also inhibits the inflammatory process (Yamauchi et al., 2001; Diez and Iglesias, 2003), are decreased in obesity. This suggests that the modulation of adipocytokine secretion from adipose tissue is critical for preventing obesity-induced metabolic abnormalities. Tomato is a common and widely consumed vegetable containing various compounds with health benefits. It has been reported that regular tomato consumption decreases the incidence of cardiovascular diseases (Pandey et al., 1995) and platelet aggregation in type 2 diabetes (Lazarus et al., 2004). Naringenin chalcone is one of the predominant flavonoids found in tomatoes, and accumulates almost exclusively in the tomato peel. This flavonoid is an intermediate in the biosynthetic pathway for flavonols and is converted to naringenin by chalcone isomerase (Muir et al., 2001). We previously reported that naringenin chalcone suppressed allergic reactions by inhibiting histamine release (Yamamoto et al., 2004). It has also been reported that naringenin chalcone inhibits the production and secretion of TNF- ␣ and MCP-1 from macrophages (Hirai et al., 2007), and recent reports have shown that some flavonoids can modulate the secretion of adipocytokines from adipocytes (Hassan et al., 2007; Kunimasa et al., 2009). However, little is known about the regulation of gene expression in adipocytes by naringenin chalcone. The present study therefore aimed to clarify the effects of naringenin chalcone on adipocyte functions related to metabolic processes, including adipocytokine production. We examined the effects of naringenin chalcone on
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adiponectin production and the changes in patterns of gene expression using DNA microarray analysis of 3T3-L1 adipocytes. 2. Materials and methods
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to the manufacturer’s instructions. Twenty-four hours after transfection, the transfected cells were cultured in medium containing the indicated concentrations of naringenin chalcone for an additional 24 h. Luciferase reporter activities were quantified using the dual luciferase system (Promega, WI, USA), in accordance with the manufacturer’s protocols.
2.1. Materials 2.7. DNA microarray analysis Naringenin chalcone was prepared from tomato peel (Yamamoto et al., 2004). Briefly, tomato extracts containing polyphenols were extracted from tomato peel with 60% (v/v) ethanol at 60 ◦ C for 2 h. Subsequently, naringenin chalcone was purified from the crude extracts by high performance liquid chromatography fractionation, and was dissolved in dimethyl sulfoxide to prepare stock solutions (200 mM). All the other chemicals used were from Sigma (MO, USA). 2.2. Cell culture and treatment NIH-3T3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (Invitrogen Japan, Tokyo, Japan). 3T3-L1 preadipocytes were cultured in DMEM containing 10% bovine serum (Invitrogen Japan). Two days after confluence, cells were differentiated into adipocytes by the addition of differentiation medium (DMEM containing 10% FBS, 0.5 mM 3-isobuthyl1-methylxantine, 1 M dexamethasone, and 10 g/ml insulin) in the presence or absence of the indicated concentration of naringenin chalcone (day 0). After 2 days, the 3T3-L1 cells were transferred to adipocyte-growing medium (DMEM containing 10% FBS and 5 g/ml insulin) in the presence or absence of the indicated concentrations of naringenin chalcone (25–100 M), which was replenished every 2 days. Dimethyl sulfoxide was used as a vehicle control for naringenin chalcone (0.1%). Culture medium was collected on day 8 and the amount of adiponectin secreted during the last 48 h of differentiation (days 6–8) was measured by enzyme-linked immunosorbent assay (ELISA). Total RNA was extracted for quantitative reverse transcription-polymerase chain reaction (RT-PCR) and DNA microarray analysis. These experiments were performed in triplicate. 2.3. Measurement of adiponectin production The concentration of adiponectin in the culture medium was determined by ELISA. ELISAs were conducted using adiponectin ELISA kits (Otsuka, Tokyo, Japan). 2.4. RNA extraction Total RNA was isolated from untreated control and treated cells using TRIzol (Invitrogen Japan) and purified using an RNeasy mini Kit (Qiagen, Hilden, Germany). Only samples with a quality ratio of 1.6–2.0 measured at A260 nm/A280 nm were used for quantitative RT-PCR and DNA microarray experiments. 2.5. Quantitative RT-PCR Changes in the expression of the adiponectin and AdipoR2 gene were assessed using a SYBR® PrimeScript® RT-PCR Kit (Takara-Bio, Shiga, Japan), according to the manufacturer’s instructions. Briefly, total RNA was reverse-transcribed into complementary DNA using a PrimeScript® RT reagent Kit (Takara-Bio). Subsequent real-time quantitative RT-PCR was performed with SYBR® Premix Ex TaqTM (TakaraBio), with PCR amplification carried out for 1 cycle at 95 ◦ C for 10 s, then 45 cycles at 95 ◦ C for 5 s and at 60 ◦ C for 20 s. Subsequently, a dissociation curve was generated to ensure amplification of only the proper PCR product. All real-time reactions were carried out on an MxPro 3000P QPCR System (Stratagene, CA, USA), and analysis was done using MxPro software (Stratagene). Relative expression levels were calculated according to standard curves obtained from real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. The primer sequences used were: adiponectin 5 -GAT GGC AGA GAT GGC ACT CC-3 (forward) and 5 -CTT GCC AGT GCT GCC GTC AT-3 (reverse), AdipoR2 5 -CTG TAA CCC ACA ACC TTG CT -3 (forward) and 5 -GGA ACC CTT CTG AGA TGA CA-3 (reverse), cyclophilin 5 -TGG TGA CTT TAC ACG CCA TA-3 (forward) and 5 -CAG TCT TGG CAG TGC AGA TA-3 (reverse). 2.6. Luciferase reporter assay A luciferase reporter assay was performed as described previously (Arimura et al., 2004; Kanayama et al., 2007). Briefly, in assays to measure the transcription of the adiponectin and perilipin genes, pGL4-adiponectin (a luciferase reporter plasmid containing about 1000 bp of the adiponectin 5 -flanking region) or pGL3perilipin (a luciferase reporter plasmid containing the segment around −2000 bp of the perilipin 5 -flanking region), pCMX-PPAR␥ (an expression plasmid for peroxisome proliferator-activated receptor ␥, PPAR␥), and pRL-CMV (an internal control plasmid) were transfected into NIH-3T3 cells cultured on 12-well plates. In assays using the GAL4-PPAR␥ chimeric protein, pG5-Luc (a reporter plasmid containing consensus Gal4-binding sites), pM-PPAR␥ (an expression plasmid for a chimeric protein for a GAL4 DNA-binding domain and PPAR␥ ligand-binding domain), and pRL-CMV were transfected into NIH-3T3 cells cultured on 12-well plates. Transfections were performed using Lipofectamine 2000 (Invitrogen Japan), according
DNA microarray analysis was performed using Mouse Genome 430 2.0 Array (Affymetrix Santa Clara, CA, USA), according to the manufacturer’s protocol. Briefly, 2 g of total RNA was reverse-transcribed into cDNA using a poly(dT) oligonucleotide attached to a T7 promoter and converted to dsDNA, which was then used as a template for the synthesis of biotinylated cRNA by T7 RNA polymerase. Labeled cRNA was fragmented and hybridized to the array at 45 ◦ C for 16 h. After hybridization, the array was washed and stained with streptavidin-phycoerythrin. Fluorescent signals were scanned using the Affymetrix GeneChip System. 2.8. Analysis of DNA microarray data Affymetrix GCOS software was used to reduce the array images to the intensity of each probe (CEL files). The CEL files were quantified using the DFW algorithm (Chen et al., 2007) using the statistical language R (Ihaka and Gentleman, 1996) and Bioconductor (Gentleman et al., 2004). The differentially expressed genes were identified using Rank Products (Breitling et al., 2004). The annotation file for the Mouse Genome 430 2.0 Array was downloaded from the Affymetrix website. DAVID was used to detect over-represented Gene Ontology (GO) categories in each group of differentially expressed genes (DEGs) (Dennis et al., 2003; Huang da et al., 2009). We compared the DEGs with the Mouse Genome 430 2.0 Array background. The functional annotation chart, a tool integrated in DAVID, was applied to examine significantly over-represented GO categories. The Benjamini and Hochberg FDR correction (Benjamini and Hochberg, 1995) was employed to correct for multiple testing. An FDR-corrected p value < 0.01 was accepted as significant. 2.9. Statistical analysis All values were represented as means ± standard deviations. Unpaired Student’s t-tests were used to analyze differences between control and treated cells. One-way analysis of variance (ANOVA) was used for experiments with three or more groups, followed by Fisher’s protected least significant difference test, with p < 0.05 accepted as significant.
3. Results 3.1. Naringenin chalcone enhances the genetic expression of adiponectin and promotes adiponectin secretion from 3T3-L1 adipocytes We first examined the effects of naringenin chalcone on the expression and production of adiponectin in cultured 3T3L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes in the presence or absence of the indicated concentrations of naringenin chalcone for 8 days. Naringenin chalcone significantly increased adiponectin mRNA levels up to 8.0-fold in a dose-dependent manner (p < 0.001) (Fig. 1A). The results of ELISAs showed that the amount of adiponectin secreted during the last 48 h of differentiation (days 6–8) was also increased (up to 2.2-fold, p < 0.001) by naringenin chalcone treatment (Fig. 1B). 3.2. Naringenin chalcone stimulates the activity of PPAR We carried out luciferase assays using a reporter gene including the adiponectin 5 -flanking region, which contains the PPARresponsive element (PPRE), to examine if naringenin chalcone stimulated the transcription of the adiponectin gene. NIH-3T3 cells were co-transfected with the adiponectin reporter plasmid and the expression plasmid for PPAR␥, which plays critical roles in the transcriptional activation of adipocytokines including adiponectin (Lehmann et al., 1995; Combs et al., 2002). The cells were cultured with naringenin chalcone for the last 24 h. Naringenin chalcone dose-dependently enhanced luciferase activity by up to 1.5-fold (p < 0.01) (Fig. 2A). The same results were obtained using the perilipin promoter reporter plasmid (up to 1.4-fold, p < 0.01), which also contains a functional PPRE (Arimura et al., 2004) (Fig. 2B).
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Fig. 1. Naringenin chalcone induces adiponectin expression. 3T3-L1 cells were treated with naringenin chalcone at the indicated concentration throughout a period of differentiation (8 days). (A) Total RNA was prepared 8 days after differentiation induction. Relative mRNA levels of adiponectin were calculated according to standard curves obtained by real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. (B) Adiponectin secretion levels during the last 48 h of differentiation. Six days after differentiation induction, 3T3-L1 adipocytes were incubated in medium containing 10% FBS and the indicated dose of naringenin chalcone for 48 h, and the medium was then collected to measure the amount of secreted adiponectin. The control value was taken as 100%. The data are shown as the means ± S.D. of triplicate cultures.
Fig. 2. Naringenin chalcone stimulates PPAR␥-mediated transcription. NIH-3T3 cells were transfected with one of the reporter plasmids for adiponectin (A) and perilipin (B) together with pRL-CMV (an internal control plasmid) and expression plasmid for PPAR␥. (C) NIH-3T3 cells were transfected with pG5-Luc (a reporter plasmid containing consensus Gal4-binding sites), pM-PPAR␥ (an expression plasmid for a fusion protein, the Gal4 DNA-binding domain and PPAR␥ ligand-binding domain), and pRL-CMV. Twenty-four hours after transfection, cells were treated with naringenin chalcone at the indicated concentration for 24 h. Luciferase reporter activities were then quantified. The control value was taken as unity. The data are shown as the means ± S.D. of triplicate cultures.
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Table 1 Significantly enriched GO terms (metabolic process and its child terms) found in top 1153 up-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
These data indicate that naringenin chalcone augmented the genetic expression of PPAR␥ targets. To confirm the direct effect of naringenin chalcone on the transcriptional activity of PPAR␥, we employed a heterologous Gal4 system. In this assay, luciferase gene transcription is driven by a promoter containing consensus Gal4-binding sites, with no co-regulatory transcription factors. NIH-3T3 cells were transfected with a reporter plasmid (pG5-Luc) and the expression plasmid encoding the PPAR␥ ligand-binding domain coupled to the DNA-binding domain of Gal4 (pM-PPAR␥). Naringenin chalcone significantly enhanced the transcription of the reporter gene in a dose-dependent manner (up to 1.8-fold, p < 0.001) (Fig. 2C). These results indicate that naringenin chalcone accelerated the transcription of adiponectin by stimulating the transcriptional activity of PPAR␥. 3.3. DNA microarray analysis and GO on up- or down-regulated genes We performed DNA microarray experiments to explore the effects of naringenin chalcone on adipocyte functions. By applying R statistical software (Ihaka and Gentleman, 1996), we found that 1153 transcripts were up-regulated and 1031 transcripts were down-regulated in naringenin chalcone-treated adipocytes, compared with control, untreated adipocytes (FDR < 0.05). Using the DAVID bioinformatics resources (Dennis et al., 2003; Huang da et al., 2009), we compared the gene lists mentioned above with the Mouse Genome 430 2.0 Array background. To extract statistically over-represented categories from the gene lists, we applied the DAVID functional annotation chart tool. The relationships between significantly enriched categories are shown in supplemental Fig.
S1. In the hierarchical structure of GO, the more specific categories appear in the deeper hierarchy. Categories with more important biological meanings therefore appear at the lower end of the graph. The significantly enriched categories of genes that were up-regulated by naringenin chalcone treatment are summarized in Tables 1 and 2. Genes related to mitochondrial energy production (‘ubiquinone biosynthetic process’, ‘mitochondrial electron transport, NADH to ubiquinone’, ‘ATP synthesis coupled proton transport’, ‘mitochondrial membrane organization’) were significantly up-regulated in the naringenin chalcone-treated adipocytes (p = 2.0E-03, 7.3E-05, 5.0E-03, and 1.1E-03, respectively). There was also significant up-regulation of genes associated with ‘glucose catabolic process’, ‘fatty acid beta-oxidation’ and ‘tricarboxylic acid cycle’ (p = 2.0E-03, 3.7E-03, and 4.4E-14, respectively). These observations suggest that naringenin chalcone enhances energy production by stimulating glucose utilization, fatty acid oxidation, and oxidative phosphorylation in adipocytes. Gene categories related to ‘cell adhesion’, ‘development’, and ‘morphogenesis’ were conversely down-regulated by naringenin chalcone treatment (Table 3). The results of this microarray experiment also confirmed the significant up-regulation of adiponectin transcripts in naringenin chalcone-treated adipocytes. Naringenin chalcone treatment also stimulated the genetic expression of one of the adiponectin receptors, AdipoR2. The results of quantitative RT-PCR analysis confirmed significant up-regulation of AdipoR2 expression (1.8-fold, p < 0.01) (Fig. 3). These results suggest that naringenin chalcone is capable of enhancing adiponectin functions in adipocytes via both increased adiponectin secretion and increased receptor expression.
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T. Horiba et al. / Molecular and Cellular Endocrinology 323 (2010) 208–214 Table 2 Significantly enriched GO terms (cellular process and its child terms) found in top 1153 up-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
Table 3 Significantly enriched GO terms found in top 1031 down-regulated genes during 100 M naringenin chalcone treatment (p < 0.01). FDR-corrected p values of the categories appearing in the deepest hierarchy are shadowed.
4. Discussion
Fig. 3. Naringenin chalcone enhances AdipoR2 expression. 3T3-L1 cells were treated with or without 100 M naringenin chalcone throughout a period of differentiation (8 days). Total RNA was prepared 8 days after differentiation induction. Relative mRNA levels of AdipoR2 were calculated according to standard curves obtained by real-time PCR using serial dilutions of templates, and normalized to the housekeeping gene cyclophilin. The control value was taken as 100%. The data are shown as the means ± S.D. of triplicate cultures.
The current study found that naringenin chalcone enhanced adiponectin gene expression and its secretion from 3T3-L1 adipocytes. It is well established that adiponectin gene expression is regulated by PPAR␥, and that a functional PPRE is present in the 5 -flanking promoter region of the adiponectin gene (Iwaki et al., 2003). PPAR␥ has been shown to be one of key regulators of adipocyte differentiation, and compounds such as thiazolidinediones stimulate PPAR␥ transcriptional activity and improve adipocyte functions by increasing the expression levels of genes associated with lipid metabolism (Ferre, 2004). The results of luciferase reporter gene assays in this study confirmed that naringenin chalcone enhanced PPAR␥-mediated transcription of both the adiponectin and perilipin genes. Consistent with enhanced PPAR␥ transcriptional activity, naringenin chalcone promoted differentiation of adipocytes, assessed by triglyceride accumulation, with no notable toxicity at concentrations up to 100 M (data not shown). It is likely that the increase in adiponectin gene expression in 3T3-L1 adipocytes was responsible for the elevated secretion of adiponectin into the culture medium. Plasma adiponectin levels were increased when naringenin chalcone was orally administered
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to diabetic KK-Ay mice in our preliminary experiments (data not shown). In addition, naringenin chalcone has been reported to have inhibitory effects on the secretion of TNF␣- and MCP-1, which are associated with induction of insulin resistance (Hirai et al., 2007). Overall, these results suggest that naringenin chalcone is capable of enhancing insulin sensitivity via modulating the expression and secretion of various adipocytokines in adipose tissues. DNA microarray analysis revealed that various clusters of genes related to energy metabolism were simultaneously up-regulated, whilst a few categories related to functions such as cell adhesion and developmental processes were down-regulated by naringenin chalcone treatment. It has previously been reported that the expression of genes related to cell adhesion were down-regulated by the activation of PPAR␥ (Galli et al., 2002). Thus, it is possible that naringenin chalcone could exert certain physiological effects due to the reduced expression of these genes, in addition to its effects due to increased adiponectin gene expression. Interestingly, the expression of genes involved in oxidative phosphorylation was markedly augmented in naringenin chalconetreated adipocytes. The oxidative phosphorylation pathway is the process by which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers. This process takes place in mitochondria and is the major source of the ATP supply in aerobic organisms. Previous studies have reported that mitochondrial activity is impaired (Morino et al., 2005; Kelley et al., 2002; Petersen et al., 2005) and that the mRNA levels of genes associated with mitochondrial oxidative metabolism are reduced in skeletal muscle of insulin-resistant subjects (Mootha et al., 2003; Patti et al., 2003). Another report demonstrated a significant reduction in the expression of genes encoding components of mitochondrial oxidative phosphorylation in adipose tissue from obese monozygotic twins (Mustelin et al., 2008). These results indicate that mitochondrial dysfunction and defects in expression of genes involved in oxidative phosphorylation are characteristic of insulin resistance. Our current results suggest the possibility that naringenin chalcone is capable of enhancing insulin sensitivity by improving mitochondrial energy metabolism in adipocytes. Two types of cell surface receptors for adiponectin, AdipoR1 and AdipoR2, have so far been identified (Yamauchi et al., 2003). AdipoR1 has been identified primarily in skeletal muscles, while AdipoR2 is most abundant in the liver in mice. Expression of AdipoR receptors in mouse adipose tissue has also been reported, with a decreased expression of these receptors in insulin-resistant mice (Tsuchida et al., 2004). The results of our microarray and quantitative RT-PCR experiments indicated that the genetic expression of AdipoR2 was enhanced in naringenin chalcone-treated adipocytes, suggesting that naringenin chalcone affects adipocyte functions by stimulating the autocrine and/or paracrine effects of adiponectin. This model could account for the observed increase in expression of genes involved in fatty acid beta-oxidation in naringenin chalcone-treated adipocytes. Since the genetic expression of AdipoR in adipose tissue is not regulated by thiazolidinediones (Li et al., 2007), the increased expression of AdipoR2 by naringenin chalcone is unlikely to be mediated through PPAR␥ activation. The molecular mechanisms responsible for the enhancement of AdipoR2 gene expression by naringenin chalcone is now under investigation. Recent studies have uncovered relationships between caloric restriction, adipocytokine regulation, and the regulation of lipid metabolism. Caloric restriction increases plasma adiponectin and improves lipid metabolism in growth hormone transgenic mice (Wang et al., 2007). Another study demonstrated that caloric restriction significantly increased adipose tissue contents of adiponectin, thereby stimulating the expression of genes involved in fatty acid oxidation and ameliorating insulin resistance (Zhu et al., 2007). Many of the changes in gene expression mentioned above occurred in naringenin chalcone-treated adipocytes in our
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study. Although our study was limited by the use of an adipocyte cell line, rather than adipose tissues from animals fed naringenin chalcone, the results suggest that naringenin chalcone exerts beneficial effects on adipocyte functions by mimicking the effects of caloric restriction in adipose tissue. Naringenin chalcone has been detected as a glucuronide conjugate in rat plasma (Yoshimura et al., 2009), while another study demonstrated that flavonoid metabolites could be deconjugated during inflammation, and contribute to its aglycone bioactivity (Kawai et al., 2008). Overall, these results suggest that orally administered naringenin chalcone is capable of altering gene expression in adipose tissue as follows: (1) Naringenin chalcone is absorbed and metabolized into glucuronide conjugate; (2) it is delivered to the target sites, such as white adipose tissues; (3) it is converted to aglycone, and regulates gene expression. However, fasting has been shown to augment hepatic expression of AdipoR2 in young pigs (Liu et al., 2008). Considering the up-regulation of AdipoR2 in adipocytes by naringenin chalcone and its putative mimicking of the effects of caloric restriction, it is possible that naringenin chalcone could also affect hepatic energy metabolism. Further studies are required to elucidate the effects of naringenin chalcone on whole-body energy metabolism. In conclusion, the tomato flavonoid naringenin chalcone stimulates adiponectin gene expression in adipocytes through PPAR␥ activation. Naringenin chalcone also enhances the genetic expression of AdipoR2 in adipocytes, thereby amplifying its effects on adiponectin. The results of DNA microarray analysis indicate that transcriptional alterations in mitochondrial oxidative metabolism caused by naringenin chalcone may produce desirable insulinsensitizing effects. These results suggest that eating naringenin chalcone could contribute to the prevention and improvement of insulin resistance and related metabolic syndrome. Acknowledgments We thank Dr. A. Obata and Dr. A. Matsuyama for valuable suggestions and discussions, and Mr. T. Manaka and Mr. K. Okada for naringenin chalcone preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2010.03.020. References Arimura, N., Horiba, T., Imagawa, M., Shimizu, M., Sato, R., 2004. The peroxisome proliferator-activated receptor gamma regulates expression of the perilipin gene in adipocytes. J. Biol. Chem. 279, 10070–10076. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. 57, 289–300. Breitling, R., Armengaud, P., Amtmann, A., Herzyk, P., 2004. Rank products: a simple, yet powerful, new method to detect differentially regulated genes in replicated microarray experiments. FEBS Lett. 573, 83–92. Chen, Z., McGee, M., Liu, Q., Scheuermann, R.H., 2007. A distribution free summarization method for Affymetrix GeneChip arrays. Bioinformatics 23, 321–327. Combs, T.P., Wagner, J.A., Berger, J., Doebber, T., Wang, W.J., Zhang, B.B., Tanen, M., Berg, A.H., O’Rahilly, S., Savage, D.B., Chatterjee, K., Weiss, S., Larson, P.J., Gottesdiener, K.M., Gertz, B.J., Charron, M.J., Scherer, P.E., Moller, D.E., 2002. Induction of adipocyte complement-related protein of 30 kilodaltons by PPARgamma agonists: a potential mechanism of insulin sensitization. Endocrinology 143, 998–1007. Dennis Jr., G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., Lempicki, R.A., 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome Biol. 4, P3. Diez, J.J., Iglesias, P., 2003. The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur. J. Endocrinol. 148, 293–300. Fasshauer, M., Paschke, R., 2003. Regulation of adipocytokines and insulin resistance. Diabetologia 46, 1594–1603. Ferre, P., 2004. The biology of peroxisome proliferator-activated receptors: relationship with lipid metabolism and insulin sensitivity. Diabetes 53 (Suppl. 1), S43–S50.
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