Rutaecarpine Analogues Reduce Lipid Accumulation in Adipocytes

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Rutaecarpine Analogues Reduce Lipid Accumulation in Adipocytes via Inhibiting Adipogenesis/Lipogenesis with AMPK Activation and UPR Suppression Ying-Chun Chen,† Xiao-Yi Zeng,‡ Yan He,† Hong Liu,† Bin Wang,† Han Zhou,† Jian-Wen Chen,† Pei-Qing Liu,† Lian-Quan Gu,† Ji-Ming Ye,*,‡ and Zhi-Shu Huang*,† †

School of Pharmaceutical Sciences and Institute of Medicinal Chemistry, Sun Yat-sen University, Guangzhou, China Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of Health Sciences, RMIT University, Melbourne, Victoria, Australia

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S Supporting Information *

ABSTRACT: Obesity is characterized by expansion of adipose tissue, which results from an increase in adipocyte number (adipogenesis) and adipocyte size (lipogenesis). A reversal of these processes has been suggested to be a potential antiobetic therapy. Rutaecarpine (Rut) and its novel analogues (R17 and R18) were identified to exert potent effect in reducing lipid accumulation during adipocyte differentiation in 3T3-L1 adipocytes with little cytotoxicity. All three compounds reduced lipid accumulation in a dose-dependent manner, while R17 and R18 exhibited much more potent inhibitory effects compared to that of Rut. Further studies showed that R17 suppressed both adipogenesis and lipogenesis during all stages of adipocyte differentiation as indicated by the reduced protein and mRNA levels of key regulators of adipogenesis/lipogenesis, including PPARγ, C/EBPα, SREBP-1c, ACC, FAS, and SCD-1. We next examined the effect of R17 on the UPR pathway and the results showed that the UPR markers (PERK, eIF2α, IRE1α, and spliced XBP1 mRNA) were all significantly reduced by R17. Further studies revealed that R17 persistently activated AMPK during differentiation, suggesting that the AMPK may be an upstream mechanism for the effect of R17 on adipogenesis and lipogenesis via the adipogenic/ lipogenic markers and the UPR pathway. Finally, studies in fast/refeeding mice demonstrated that R17 administration was able to reduce epididymal fat mass and the levels of plasma TG and FFA in vivo. Our results suggest that rutaecarpine analogues may have therapeutic potential for obesity and related metabolic disorders. The mechanism involves the suppression of adipogenic/ lipogenic proteins and the suppression of the UPR pathway possibly via the AMPK.

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induce the expression of C/EBPα and peroxisome proliferatoractivated receptor γ (PPARγ), which cross-regulate to facilitate the formation of mature adipocytes and promote lipid production.7 PPARγ is viewed as a master regulator of adipogenesis, as overexpression of PPARγ is sufficient to induce adipogenesis in the absence of extracellular hormones and, on the other hand, PPARγ-deficiency prevents precursor cells from differentiating into mature adipocytes.6 In addition, sterol regulatory element-binding protein 1c (SREBP-1c) is also a master regulator of adipogenesis and lipogenesis by regulating the expression of lipogenic proteins including acetylCoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD-1).5,8,9 The endoplasmic reticulum (ER) is a central cellular organelle playing a crucial role in protein folding, assembly,

besity, a medical condition affecting 10% of the world’s adult population, is a major risk factor contributing to a number of metabolic abnormalities including type 2 diabetes, hypertension, and atherosclerosis.1 Obesity is characterized by increased mass of white adipose tissue, which stores excess energy in the form of triglyceride (TG). The expansion of adipose tissue results from an increase in adipocyte number (due to increased fat cell differentiation, namely, adipogenesis) and adipocyte size (partly due to increased lipid production, namely, lipogenesis).2,3 In addition to the increase of fatty acid oxidation, e.g., through exercise, a number of recent studies suggested the inhibition of adipogenesis and lipogenesis would be beneficial to prevent the initiation and progression of obesity.2−4 The differentiation of preadipocytes and the subsequent lipid production in mature adipocytes are controlled by orchestrated expressions of a number of transcription factors.5,6 At the early stage of preadipocyte differentiation, the transient expression of CCAAT/enhancer binding protein-β (C/EBPβ) and C/EBPδ © XXXX American Chemical Society

Received: June 1, 2013 Accepted: August 20, 2013

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dx.doi.org/10.1021/cb4003893 | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

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Figure 1. Identification of Rut and its analogues (R17 and R18) as inhibitors of adipogenesis via 3T3-L1 cell-based screening. 3T3-L1 preadipocytes were treated with 10 μM compounds on day 0 for 3 days during the differentiation. Then the lipid content was evaluated by TG assay on day 6. Berberine (10 μM) was used as the positive control. (A) Overview of the numbers of compounds selected from different classes according to their chemical structures. Ruts, rutaecarpine derivatives; BBRs, berberine derivatives; Mansonones, mansonone derivatives; Curcumines, curcumine derivatives. (B) Representative screening results of Rut and its analogues. Purple bars indicate screening hits (compounds reducing triglyceride level by >50%). (C) Chemical structures of Rut, R17, and R18. (D) Synthesis of target compounds R17 and R18. Reagents and conditions: (a) triethyl orthopropionate, reflux; (b) Br2, NaAc, HAc, 60 °C; (c) PhNHNH2, EtOH, reflux; (d) PPA, 150 °C; (e) POCl3, DMF, N2, rt; (f) NH2(CH2)3N(CH3)2 or NH2(CH2)3N(C2H5)2, triethylamine, methylbenzene, reflux.

rutaecarpine on reducing food intake by suppressing the expression of NPY and AgRP.18 Therefore, to identify novel antiobesity compounds, we investigated the antiobesity properties of Rut and its analogues R17 and R18 (Figure 1C) by evaluating their effects on lipid accumulation using 3T3-L1 cells. Our results demonstrated that R17 displayed the most potent effects on lipid accumulation compared with the other two compounds with little cytotoxicity. Consistent with its potent lipid-lowering effect, R17 suppressed a panel of key adipogenesis/lipogenesis regulators (C/EBPβ, C/EBPδ, C/ EBPα, PPARγ, and SREBP-1c) and their downstream lipogenic enzymes (ACC, FAS, and SCD-1), with a concurrent downregulation of the protein expression of three key proteins (PERK, eIF2α, and IRE1) in the UPR pathway, which was reported to be implicated in the regulation of adipogenesis and lipogenesis. Furthermore, we have identified the activation of the AMPK pathway as a likely mechanism underlying the antiobesity effect of R17. Finally, administration of R17 in fast/ refeeding mice significantly reduced epididymal fat mass and plasma levels of TG and free fatty acid (FFA). This is the first time that Rut and its analogues are reported to inhibit

and secretion. ER stress occurs when there is overproduction of proteins or accumulation of misfolded proteins in the ER lumen.10,11 During ER stress, the unfolded protein response (UPR) is activated to restore ER homeostasis. The UPR pathway is composed of three signaling branches initiated by three ER transmembrane protein sensors, inositol requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6).10,11 Upon ER stress, the activation of PERK phosphorylates the eukaryotic translation initiation factor 2α (eIF2α), while activated IRE1α cleaves a 26bp segment out of the XBP1 mRNA, creating a spliced mRNA that translates an active form of the transcription factor.12,13 Recent studies have shown that an activation of the UPR pathway is closely linked to adipogenesis via regulating the transcription factor SREBP1c.13−16 Rutaecarpine (Rut, Figure 1C), an alkaloid isolated from Evodia rutaecarpa and related herbs, has shown a variety of intriguing biological properties such as antithrombotic, anticancer, anti-inflammatory, and analgesic activities.17 However, there is little report concerning the antiobesity effect of rutaecarpine and its analogues, apart from the effect of B



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adipogenesis and lipogenesis, demonstrating that rutaecarpine analogues could be a novel and promising class of compounds for the treatment of obesity. In addition, this is the first observation showing a suppression of the UPR pathway associated with AMPK by rutaecarpines as a mechanism for reducing cellular lipid accumulation, providing a proof of concept for targeting these pathways for identification of novel antiobetic compounds.

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RESULTS AND DISCUSSION Identification of Rut and Its Analogues (R17 and R18) as Inhibitors of Adipogenesis via 3T3-L1 Cell-Based Screening. The 3T3-L1 cell line recapitulates different stages of preadipocyte differentiation and is a well-established in vitro model for the metabolic study of obesity.6,19 3T3-L1 cell-based screening for compounds with lipid-lowering properties has been shown to be an efficacious tool for the identification of new antiobesity and antidiabetic compounds.20 Using this approach, we then proceeded to screen our compound library, which contains natural products and related derivatives with diverse structures. Out of 155 compounds from different classes (Figure 1A) screened at a concentration of 10 μM, 9 hits (compounds with efficacy in reducing triglyceride accumulation by >50%) were found in the class of Rut (Figure 1B). The class of Rut comprises Rut and 34 rutaecarpine derivatives, which were synthesized by introducing side chain(s) or adjusting the ring size of rutaecarpine to improve biological activities (common strategies in the modification of natural products). Among them, the natural product Rut reduced lipid accumulation by ∼60%. Most notably, R17 and R18 (Figure 1C), two novel small molecules developed from Rut by attaching a side chain with terminal amino group and shrinking the ring size from six to five, displayed the most profound lipidlowering effect (97.4 ± 2.8% and 91.9 ± 9.0%, respectively, Figure 1B), without observed toxic effects. The synthetic route of R17 and R18 is described in Figure 1D. This is the first time that rutaecarpine and its analogues were reported to suppress lipid accumulation in cells. Comparison of the Inhibitory Effects on Lipid Accumulation in 3T3-L1 Cells. To further evaluate the inhibitory effects of Rut and its analogues (R17 and R18) on lipid accumulation, 2 day postconfluent 3T3-L1 preadipocytes were treated with Rut, R18, or R17 at indicated concentrations for 6 days after the initiation of differentiation. On day 6, intracellular lipid accumulation was determined by a TG assay. All three compounds decreased intracellular lipid accumulation in a dose-dependent manner in 3T3-L1 cells (Figure 2A). These lipid-lowering effects were further confirmed by Oil Red O staining as shown in Figure 2B. At the maximum dosage used (3 μM), R17 and R18 reduced the lipid content by up to ∼90% and ∼70% (relative to vehicle-treated control cells), respectively, while Rut at the same concentration achieved only ∼30% reduction (Figure 2A). Furthermore, R17 and R18 reduced the lipid accumulation in 3T3-L1 adipocytes more effectively (EC50 = 0.34 ± 0.10 and 1.90 ± 0.21 μM, respectively) compared to Rut (EC50 = 8.48 ± 1.27 μM). In addition, the lipid-lowering effect of Rut and its analogues (R17 and R18) is not cellspecific, as they also dose-dependently suppressed high fructose-induced lipid accumulation in FAO hepatoma cells (Supplementary Figure 1). To exclude the possibility that the reduction in lipid accumulation was due to cytotoxicity imposed by Rut or its analogues R17 and R18, an LDH release/cytotoxicity assay was

Figure 2. Effects of Rut and its analogues (R17 and R18) on lipid accumulation in 3T3-L1 cells. 3T3-L1 preadipocytes were treated with compounds at indicated concentration for 6 days during the differentiation. Then the lipid content was evaluated by TG assay, and the morphological change was visualized by microscopy imaging at 10X magnification. (A) Dose−response curve of Rut, R17, and R18. To make the logarithmic graph, 0 μM was set as 0.0001 μM. Data are expressed as the mean ± SE of 3 independent experiments. The symbols *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 vs control, respectively. (B) Lipid accumulation of 3T3-L1 adipocytes treated with indicated compounds was visualized by Oil Red O staining, and pictures were taken on day 6 with 10X magnification. UD: undifferentiated cells. Control: differentiated cells without compounds. (C) Rut, R17, and R18 treatment is not cytotoxic to 3T3-L1 cells. Confluent 3T3-L1 cells were treated with 3 or 10 μM Rut, R17, or R18 for up to 6 days during preadipocyte differentiation as described in Methods. Medium was collected, and LDH release was measured. Data are expressed as the mean ± SE of 3 independent experiments. NS indicates no significant vs control.

performed during 3T3-L1 preadipocyte differentiation in the presence of Rut, R17, or R18 for 6 days. As shown in Figure 2C, neither Rut nor its analogues caused detectable cytotoxicity at a concentration of 3 or 10 μM. We further examined the toxic effect of these compounds in HEK293 cells, which are C



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Figure 3. Effects of R17 at various intervals during adipocyte differentiation. (A) Drug treatment schematic diagram. 3T3-L1 preadipocytes were treated with 3 μM R17 for various periods. On day 9, intracellular lipid accumulation was determined by a TG assay. (B) Lipid accumulation determined by TG assays. Data are expressed as the mean ± SE of 3 independent experiments. The symbols *, ** and *** indicate significant differences at P < 0.05, P < 0.01 and P < 0.001 vs control, respectively. (C) Lipid accumulation of 3T3-L1 adipocytes was visualized by Oil Red O staining and pictures were taken on day 9 with 10X magnification. UD: undifferentiated cells. Control: differentiated cells without compounds.

droplets in a similar trend as shown in representative microscopy images (Figure 3C). These data suggested rutaecarpine analogue R17 reduced lipid accumulation by inhibiting both adipogenesis and lipogenesis. R17 Inhibits Expression of Adipogenesis-Related Transcription Factors and Downstream Targets Lipogenic Enzymes. Transcription factors C/EBPβ, C/EBPδ, C/ EBPα, PPARγ, and SREBP-1c are critical regulators for adipogenesis and lipogenesis.5,6 To determine whether reduced lipid accumulation in adipocytes was due to alterations in the expression of these transcription factors, we performed RTPCR and Western blotting analysis on 3T3-L1 cells with Rut or R17 treatment at different time points. As shown in Figure 4A, both R17 and Rut decreased the mRNA levels of C/EBPβ and C/EBPδ within the first 24 h of differentiation. Then we examined effects of R17 on the mRNA levels of PPARγ C/ EBPα and SREBP1c, which are downstream targets of C/EBPβ and C/EBPδ. Consistently, R17 reduced mRNA levels of these three transcription factors compared to the control (Figure 4B) within the first 24 h of differentiation. Accordingly, the protein levels of PPARγ, C/EBPα, and SREBP-1c were also attenuated in a dose-dependent manner with R17 treatment compared to the control, supporting the RT-PCR data (Figure 4C). PPARγ, SREBP-1c, and C/EBPα are known to be regulated in early stage during adipocyte differentiation, which can

commonly used normal cells for the MTT assay. The result showed that Rut and its analogues (R17 and R18) at 3 μM did not affect cell viability compared to the vehicle control (Supplementary Figure 2). Taken together, these results suggested that the Rut analogue R17 is more efficacious in lowering lipid accumulation compared to its parent compound Rut and analogue R18 with little cytotoxicity. Therefore, R17 was chosen for the following mechanistic study. R17 Blocks Lipid Accumulation during All Stages of Adipocyte Differentiation. To determine whether R17 reduces lipid accumulation by suppressing adipogenesis and/ or lipogenesis, we treated 3T3-L1 cells with R17 for various periods, namely, days 0−3, 3−6, 6−9, 0−6, 3−6, and 0−9, which represent different stages of 3T3-L1 adipocyte differentiation (Figure 3A). On day 9, intracellular lipid content was determined by a TG assay and representative microscopy images were captured. R17 treatment during days 0−3, 3−6. and 6−9 resulted in >80%, ∼60%, and ∼40% reduction in lipid accumulation, respectively (Figure 3B). The lipid-lowering effect of R17 treatment displayed a time-dependent manner, as the 9-day treatment (days 0−9) led to greater reduction of lipid accumulation compared with the effects after 3 or 6 days of treatment. Consistent with the results on lipid accumulation, incubation of 3T3-L1 with R17 for various treatment periods led to reductions of mature adipocyte number and lipid D



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Figure 4. Effects of R17 and its parent compound Rut on adipogenesis-related transcription factors and downstream lipogenic enzymes. 3T3-L1 preadipocytes were treated with R17 or Rut for indicated periods during adipocyte differentiation and then harvested. The protein and mRNA levels were determined by Western blot and RT-PCR. (A) mRNA levels of C/EBPβ and C/EBPδ (24 h treatment). (B) mRNA levels of PPARγ, C/EBPα, and SREBP-1c (24 h treatment). (C) Protein levels of PPARγ, C/EBPα, and SREBP-1c. (3 days treatment). (D) Protein levels of ACC, FAS, and SCD-1 (6 days treatment). All data were normalized to α-tubulin, and the fold changes in expression were calculated relative to control. All data are expressed as the mean ± SE of 3 independent experiments. The symbols *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 vs control, respectively. MDI, a cocktail consisting of insulin, dexamethasone, and 1-methyl-3-isobutylxanthin. E



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Figure 5. Effects of R17 and its parent compound Rut on two branches of UPR pathway. 3T3-L1 preadipocytes were treated with R17 or Rut at indicated concentrations for 6 days during adipocyte differentiation. The protein measurements of UPR markers were examined by Western blot, and the mRNA was collected and subjected to RT-PCR to detect XBP1. (A, B) Effect of R17 and Rut on protein levels of UPR markers. All data were normalized to α-tubulin, and the fold changes in expression were calculated relative to control (control is set as 1). (C) Effect of R17 and Rut on XBP1 mRNA splicing in 3T3-L1 cells. Resultant cDNA was digested with PstI and analyzed by 3% agarose gel to visualize the activated spliced (XBP1s) and unpliced (XBP1u) products. Actin was used as a control. DTT (2 mM), an ER stress inducer, was used as a positive control. All images are from a representative of three different experiments. All data are expressed as the mean ± SE from 3 independent experiments. The symbols *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 vs control, respectively. MDI, a cocktail consisting of insulin, dexamethasone, and 1-methyl-3-isobutylxanthin.

activate the lipogenic markers such as ACC, FAS, and SCD1.9,21 These three markers are critical enzymes involved in lipogenesis. To be specific, ACC controls the synthesis of malonyl-CoA from acetyl-CoA,22 and FAS facilitates the synthesis and cytoplasmic storage of massive amounts of triglyceride.23 SCD-1 has been known to regulate the biosynthesis of unsaturated fatty acids.24 Given the reduction in lipid content and the downregulation of PPARγ, SREBP-1c and C/EBPα, we hypothesize that the activities of these lipogenic enzymes may be suppressed. As expected, the protein levels of lipogenic enzymes ACC, FAS, and SCD-1 were significantly decreased in a dose-dependent manner with the treatment of R17 for 6 days (Figure 4D). Consistent with its more potent lipid-lowering effect, 3 μM R17 almost abolished the protein expression level of these lipogenic enzymes, while the parent compound Rut at the same concentration showed similar but less potent effects on the expression of these transcription factors and lipogenic enzymes (Figure 4). As for

the other analogue R18, it also suppressed adipogenesis transcription factors and lipogenic enzymes (Supplementary Figure 3). Taken together, these findings suggest that Rut and its more potent analogue R17 suppress lipid accumulation by decreasing the expression of transcription factors and enzymes that are critical for the adipogenesis/lipogenesis. R17 Attenuates the UPR Signaling Pathway. Increasing evidence suggests that the UPR signaling pathway is involved in the regulation of adipogenesis and lipogenesis by modulating lipogenic markers such as SREBP1c, ACC, and SCD-1.13−16 A detailed study demonstrates that IRE1α activation and subsequent XBP1 mRNA splicing is indispensable for adipogenesis in 3T3-L1 cells.13 The loss of either XBP1 or IRE1α in 3T3-L1 cells or mouse embryonic fibroblasts can result in impaired adipogenesis,13−15 further supporting the crucial role of IRE1/XBP1 in adipogenesis. XBP1 is also shown to promote lipogenesis in the liver by directly modulating expression of lipogenic genes, such as SREBP1, ACC, and SCD-1.16 F



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Figure 6. Effects of R17 and its parent compound Rut on AMPK activation. 3T3-L1 preadipocytes were treated with compounds at indicated concentration for various time and then harvested. The protein levels of p/t-AMPK and p/t-ACC were determined by Western blot after treatments during (A) the first 2 h, (B) the first 8 h, or (C) day 0−6 of the differentiation. Representative images are shown. All data were normalized to β-actin, and the fold changes in expression were calculated relative to control. All data are expressed as the mean ± SE of 3 independent experiments. AICAR (0.2 mM), an AMPK activator, was used as the positive control. The symbols * (or #), ** (or ##), and *** (or ###) indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 vs control, respectively. MDI, a cocktail consisting of insulin, dexamethasone, and 1-methyl-3-isobutylxanthin.

were also upregulated during the progression of differentiation. These results are in agreement with the previous findings13,15 that showed UPR signaling pathway is activated during adipocyte differentiation. Notably, our results showed that addition of R17 markedly reduced the protein levels of these UPR markers, including the phosphorylated and total forms of PERK, eIF2α, IRE1α, as well as XBP1s and XBP1u (Figure 5A−C). These findings indicated that R17 suppressed UPR activation during adipocyte differentiation at not only phosphorylation levels but also transcription levels. DTT, an ER stress inducer,13,25 served as a positive control. For R18, it also suppressed the UPR pathway (Supplementary Figure 5). However, the parent compound Rut at the same concentration did not exhibit significant effect on these UPR markers (Figure 5A−C). Collectively, our results demonstrated the lipidlowering effect of R17 was associated with a suppression of endogenous UPR activation during adipocyte differentiation. In light of previous reports that show attenuation of UPR

Additionally, the absence of PERK in mouse embryonic fibroblasts and mammary epithelium attenuates lipogenesis and expression of genes such as SREBP1, SCD1, and FAS.14 These findings suggest that inhibition of the UPR signaling pathway may result in the suppression of adipogenesis/ lipogenesis-related pathways and thus contribute to the reduction of lipid content. Indeed, the ER stress inhibitor 4phenylbutyrate is shown to suppress adipogenesis in 3T3-L1 cells and high-fat diet-induced obesity mouse model via blocking the UPR signaling pathway.15 These data prompted us to investigate whether the suppression of UPR signaling pathway is involved in the mechanism of R17 action in adipocyte differentiation. As illustrated in Supplementary Figure 4, during adipocyte differentiation, the protein levels of both phosphorylated and total UPR markers (PERK, eIF2α, IRE1α) increased during adipocyte differentiation. Furthermore, the mRNA levels of spliced XBP1 (XBP1s), as well as unspliced XBP1 (XBP1u), G



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Figure 7. Effect of R17 administration on in vivo lipid metabolism in fast/refeeding mice. (A) Body weight, (B) food intake, (C) epididymal fat mass, (D) plasma TG, (E) plasma FFA, and (F) blood glucose. The symbols *, **, and *** indicate significant differences at P < 0.05, P < 0.01, and P < 0.001 vs vehicle goup, respectively. NS indicates no significant vs vehicle group. Data are expressed as mean ± SE (n = 10 mice/group).

activation is beneficial against adipogenesis/lipogenesis,13−16 this associated suppression of the UPR pathway may at least partially contribute to the downregulation of key lipogenic proteins and the reduction of intracellular lipids by R17. However, further studies are required to determine the precise mechanism by which R17 attenuates the UPR pathway. R17 Activates AMPK Signaling Pathway. It has been reported that the activation of AMPK leads to the inhibition of adipogenesis and lipogenesis.26−29 To investigate whether the activation of AMPK is involved in the lipid-lowering effect of R17 and Rut, the effect of R17 and Rut on the AMPK pathway was examined in 3T3-L1 cells, using AICAR, a potent AMPK activator as the positive control. Western blot analysis showed that R17 induced AMPK activation as early as the first 2 h of differentiation, as evidenced by a ∼40% increase of phosphorylation of AMPK and its downstream substrate ACC (Figure 6A). Notably, R17-induced AMPK activation was stably maintained throughout the 6-day treatment period (Figure 6B and C). Rut, at the same concentration, also induced AMPK activation, but to a lesser extent (Figure 6). All of the data indicate R17 and Rut reduce adipogenesis/ lipogenesis through activation of the AMPK pathway as an upstream mechanism. Some recent work indicated that AMPK regulates lipid synthesis in adipocytes by regulating the activity of PPARγ and C/EBPα.30,31 In addition, several reports have shown that AMPK activation suppresses ER stress, which contributes to the prevention of hypoxic injury,32 atherosclerosis,33,34 lipid-induced hepatic disorders,35 and insulin resistance.36 In addition, reduction in AMPK levels promotes ER stress.34 These findings suggest that AMPK functions as a physiological suppressor of ER stress. In this context, it is tempting to speculate that AMPK might be the upstream regulator of adipogenesis-related transcription factors and the UPR signaling pathway. Our observation that AMPK was activated within the first 2 h of differentiation (Figure 6A) or even earlier and then lasted for the 6-day treatment period, whereas the downregulation of transcription factors and UPR signaling were observed on day 3 and day 6, raise the possibility of this hypothesis. Our hypothesis (Figure 8) is supported by the very recent findings that AMPK activation can impair PPARγ activity and lipid synthesis30 and AMPK activated by

oleate prevents palmitate-induced ER stress in skeletal muscle cells.36 Further studies are required to identify the direct molecular target of the R17 and to explore whether AMPK is the upstream regulator of adipogenic transcription factors and the UPR pathway in R17 action. Effect of R17 Administration on Fast/Refeeding Mice. We next tested whether R17 was able to inhibit adipogenesis/ lipogenesis in vivo with effects on lipid metabolism in fast/ refeeding mice, a well-recognized animal model of stimulated de novo lipogenesis in adipose tissue and the liver.37−40 After 24 h of fasting, mice were given either R17 (20 mg kg−1, i.p.) or vehicle (saline) followed by 12 h of refeeding with a high fructose diet38 to stimulate de novo lipogenesis. As shown in Figure 7A−F, R17 administration significantly reduced epididymal fat mass and plasma levels of TG and FFA. These effects on lipid metabolism were independent of body weight, food intake, or blood glucose level. These results provide the in vivo evidence to indicate that R17 can inhibit adipogenesis/ lipogenesis with adipose tissue as a target site as predicted from our findings in 3T3-L1 cells Conclusions. We have demonstrated for the first time that rutaecarpine (Rut) and its novel analogues R17 and R18 potently reduce lipid accumulation in 3T3-L1 adipocytes. Specifically, R17 exerts the most potent inhibitory effects, and the underlying mechanism involves the suppression of adipogenesis-related markers and the suppression of UPR pathway associated with the activation of AMPK (Figure 8). Consistent with its effect on cultured 3T3-L1 adipocytes, R17 administration is able to reduce epididymal fat mass and plasma levels of TG and FFA in fast/refeeding mice. These findings together suggest that rutaecarpine and its analogues may represent a novel class of compounds for the prevention and treatment of obesity. In addition, our results suggest a novel phenomenon that chemical interference of the UPR activation associated with AMPK might facilitate the inhibition of adipogenesis in 3T3-L1 adipocytes, indicating targeting these pathways by small molecules may represent a potential approach for the treatment of obesity. H



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with 4 mL of ddH2O followed by filtration through a 0.22 μm filter (Millipore). Lactate Dehydrogenase (LDH) Assay. After cells were treated with compounds for 6 days, the medium and lysate were collected to measure the activity of lactate dehydrogenase (LDH) released into the culture medium from cells. The medium was collected and centrifuged at 2000 rpm at 4 °C for 10 min to remove detached cells. To measure the intracellular LDH activity, 3T3-L1 cells were lysed by two freeze− thaw cycles (chilling the plate at −80 °C for approximately 1 h followed by thawing at 37 °C for 30 min) after washing with ice-cold PBS buffer. The cell lysate was then centrifuged at 5000 rpm for 5 min, and the supernatant was ready for use. The enzyme activity in the whole cell lysate and medium was determined by a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer’s protocol. The absorbance of the samples was read at 490 nm using a Polarstar Optima microplate reader (BMG Labtech). LDH released into the medium was expressed as a percentage of the total LDH activity (equation used for LDH release (%) calculation: LDH release (%) = LDH activity in medium/(LDH activity in medium + LDH activity in lysate) × 100) as described. Western Blotting. Protein samples denatured in SDS sample buffer (125 mmol/L Tris-HCl, pH 6.8, 50% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride (Millipore) membranes. Blotted membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 for 1 h and then incubated with primary antibodies for 16 h at 4 °C. After three washes in Tris-buffered saline containing 0.1% (v/ v) Tween 20, the membranes were incubated with anti-mouse or antirabbit IgG and horseradish peroxidase-linked antibodies for 2 h. Immunoreactive signals were detected with ChemiDoc XRS Imaging System (Bio-Rad) and quantified with a Image Lab 3.0 software (BioRad). Key adipogenic markers were examined by Western blotting using specific antibodies including total- and phospho (Ser79)-acetylCoA carboxylase (ACC, Cell Signaling). Fatty acid synthase (FAS, Cell Signaling), stearoyl-CoA desaturase 1 (SCD-1, Cell Signaling), Peroxisome proliferator-activated receptor γ (PPARγ, Cell Signaling), total- and phospho (Thr172)- AMP-activated protein kinase α (AMPKα, Cell Signaling), CCAAT/enhancer-binding proteins (C/ EBPα, Santa Cruz), Sterol regulatory element- binding protein 1c (SREBP-1c, Santa Cruz). ER stress: total- and phospho (Thr980)pancreatic ER kinase (PERK, Santa Cruz), and phosphor (Ser724)inositol-requiring kinase 1α (p-IRE1α, Novus Biologicals) and the total IRE1α (IRE1α, Cell Signaling), total- and phospho (Ser51)eukaryotic translation initiation factor 2α (eIF2α, Cell Signaling). Immunolabeled bands were quantified by densitometry and representative blots are shown. Reverse-Transcription PCR. In order to conduct reversetranscription PCR (RT-PCR), total RNA was isolated, using RNAiso Plus (Takara), according to the manufacturer’s protocol. After strand cDNA was synthesized by using Oligo (dT), cDNA was used for amplification of specific target genes by PCR. β-Actin was used as the RNA loading control. PCR products were separated on 2% (w/v) agarose gels and analyzed by AlphaImager EC Imaging System (Alpha Innotech). PCR primer sequences were as follows: β-actin, sense, 5′TGGAATCCTGTGGCATCCATGAAA-3′, antisense, 5′-TAAAACGCAGCTCAGTAACAGTCC-3′; SREBP-1c, sense, 5′-CAGCTCAGAGCCGTGGTGA-3′, antisense, 5′-TGTGTGCACTTCGTAGGGTC-3′; PPARγ, sense, 5′-TGCTGTTATGGGTGAAACTCTG-3′, antisense, 5′-GAAATCAACTGTGGTAAAGGGC-3′; C/EBPα, sense, 5′-AGACATCAGCGCCTACATCG-3′, antisense, 5′CTCTTGTTTGATCACCAGCGG-3′; C/EBPβ, sense, 5′-TTATAAACCTCCCGCTCGGC-3′, antisense, 5′-CTCAGCTTGTCCACCGTCTT-3′; C/EBPδ, sense, 5′-AGCCCAACTTGGACGCCAG-3′, antisense, 5′-TCGTCGTACATGGCAGGAGT-3′. RT-PCR for XBP1 Splicing. XBP1 mRNA was obtained as described above using the specific primer set for mouse X-box binding protein (XBP-1, sense, 5′-AAACAGAGTAGCAGCGCAGACTGC-3′; antisense, 5′-GGATCTCTAAAACTAGAGGCTTGGTG-3′;) which amplifies a 601-bp cDNA product encompassing the IRE1α cleavage

Figure 8. Proposed mechamism for the effects of R17 on lipid accumulation in adipopcytes. R17 suppresses adipogenesis by activating AMPK, which leads to the downregulation of adipogenesis-related transcription factors, including C/EBPβ, C/EBPδ, PPARγ, C/EBPα, and SREBP-1, and ultimately results in decreases of downstream lipogenic enzymes, such as ACC, FAS, and SCD-1. In addition, the activation of AMPK may also lead to the suppression of the UPR signaling pathway, including the PERK/eIF2α and IRE1α/ XBP1 arm, which may at least partly contribute to the inhibition of adipogenesis. The overall downregulation of key transcription factors and proteins for adipogenesis and lipogenesis ultimately leads to a suppression of the maturation of adipocytes and a reduction of lipid production. (→, activation; |, inhibition).

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METHODS

Reagents and Chemicals. Insulin, dexamethasone, 3-isobutylxanthine (IBMX), berberine (BBR), dithiothreitol (DTT), and fructose were purchased from Sigma-Aldrich. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) was purchased from Cayman. Tissue culture reagents were obtained from Gibco. Cell Culture. 3T3-L1 preadipocytes, which are mouse embryonic fibroblasts capable of differentiating into adipocytes, were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycinglutamine (PSG) at 37 °C in 5% CO2. Adipocyte differentiation was carried out as follows: Briefly, 2 days after confluence (day 0), the medium was changed to differentiation medium (DMEM containing 10% (v/v) fetal bovine serum, 1% (v/v) PSG, 2 μg mL−1 insulin, 100 ng mL−1 dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX)). Then dexamethasone and IBMX were withdrawn after 3 days of exposure (day 3), and insulin was withdrawn after an additional 3 days (day 6). Test compounds were made as stock solutions and stored at −20 °C in aliquots. They were added on day 0 in differentiation medium (for 3 days) and replaced with another aliquot in postdifferentiation medium on day 3 (for another 3 days). 3T3-L1 cells cultured in DMEM supplemented with 0.1% (v/v) DMSO were used as a vehicle control for all experiments. Triglycerides Assay. Cells were collected at the indicated times and washed twice with ice-cold PBS buffer (0.2 M NaCl, 10 mM Na2HPO4, 3 mM KCl, 2 mM KH2PO4, pH 7.4). Then the cells were lysed, and the levels of intracellular triglycerides were determined with commercial Peridochrom TG GPO-PAP kit (Roche Diagnostics) following the manufacturer’s instruction. The results were expressed as the relative triglyceride (TG) content compared to the vehicle-treated positive control cells. Oil Red O Staining. Cells were fixed with 10% (v/v) formaldehyde in PBS for 1 h at RT and stained with freshly prepared Oil Red O working solution for 30 min followed by three washes with water. Plates were scanned using the Olympus CKX41 microscope and camera (Olympus). Oil Red O working solution was prepared by mixing 6 mL 0.35% (w/v) Oil Red O (Sigma) in isopropyl alcohol I



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sites. This fragment was further digested by PstI to reveal a restriction site that is lost after IRE1α-mediated cleavage and splicing of the mRNA. The cDNA fragments were resolved on 3% (w/v) agarose gels and analyzed by AlphaImager EC Imaging System (Alpha Innotech). Animal Study. Male C57BL/6J mice (n = 20, body weight 21 ± 2 g, 9 weeks of age) were purchased from Laboratory Animal Centre, Sun Yat-sen University, Guangzhou, China. All animal procedures were approved by the Sun Yat-sen University Committee on Ethics in the Care and Use of Laboratory Animals in accordance with the Animal Welfare Legislation of China. All animals were housed under standard conditions with free access to regular food and water. After feeding with regular diet for 1 week, they were randomly assigned to two groups (10/group). For fasting-refeeding studies, a high-fructose diet (35% (w/w) fructose and 35% (w/w) starch, HFru) diet was used to induce lipogenesis. Mice were fasted for 24 h and then given a single intraperitoneal injection of freshly prepared R17 hydrochloride (pH = 6) (20 mg kg−1, R17 group) or equal volume of saline (vehicle group). Following 12 h of refeeding with the HFru diet, the animals were sacrificed by decapitation and tissues of interest were freeze-clamped immediately. Epididymal fat mass was weighed using an analytical balance. Body weight and food intake were monitored at indicated times. Blood levels of glucose and plasma triglyceride and FFA were measured by using a portable blood test system (Synchron CX5 Systems, BECKMAN). Liver triglycerides were extracted by Folch’s method41 and determined by a triglyceride content assay kit (Beijing BHKT, China).

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary methods and results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (81273433 to Z.-S.H.), the International S&T Cooperation Program of China (2010DFA34630 to L.Q.G.), and the National Health and Medical Research Council of Australia Program Grant (535921 allocation to J.-M.Y.). The authors would like to thank J. C. Molero for his critical comments (RMIT University, Australia).

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Rutaecarpine Analogues Reduce Lipid Accumulation in Adipocytes

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