![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
, PPAR
, and Adipocyte Differentiation In Vitro1–3,
* Department of Obstetrics and Gynecology, and Department of Biostatistics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074
4 To whom correspondence should be addressed. E-mail: obgyel{at}nus.edu.sg.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
and PPAR
are used to correct dyslipidemia and to restore glycemic balance, respectively. Because the majority of diabetic patients suffer from atherogenic lipid abnormalities, in addition to insulin resistance, ligands are required that can activate both PPAR and PPAR. In this study, we used chimeric PPAR/ reporter-gene bioassays to screen herbal extracts with purported antidiabetic properties. Extracts of Astragalus membranaceus and Pueraria thomsonii significantly activated PPAR and PPAR. Bioassay-guided fractionation resulted in the isolation of the isoflavones, formononetin, and calycosin from Astragalus membranaceus, and daidzein from Pueraria thomsonii as the PPAR-activating compounds. We investigated the effects of these and 2 common isoflavones, genistein and biochanin A, using chimeric and full-length PPAR constructs in vitro. Biochanin A and formononectin were potent activators of both PPAR receptors (EC50 = 1–4 µmol/L) with PPAR/PPAR activity ratios of 1:3 in the chimeric and almost 1:1 in the full-length assay, comparable to those observed for synthetic dual PPAR-activating compounds under pharmaceutical development. There was a subtle hierarchy of PPAR/ activities, indicating that biochanin A, formononetin, and genistein were more potent than calycosin and daidzein in chimeric as well as full-length receptor assays. At low doses, only biochanin A and formononetin, but not genistein, calycosin, or daidzein, activated PPAR-driven reporter-gene activity and induced differentiation of 3T3-L1 preadipocytes. Our data suggest the potential value of isoflavones, especially biochanin A and their parent botanicals, as antidiabetic agents and for use in regulating lipid metabolism.
KEY WORDS: • PPAR • PPAR • isoflavones • adipocyte differentiation
The metabolic syndrome, wherein patients have both diabetes mellitus and dyslipidemia, is reaching epidemic proportions due to dietary factors and a sedentary lifestyle (1). A major cause of mortality in these patients is atherosclerotic macrovascular disease that results, in large part, from dyslipidemia associated with insulin-resistant diabetes. Peroxisome proliferator–activated receptors (PPAR),5 a subfamily of the 48-member steroid and nuclear-receptor superfamily, are ligand-dependent transcription factors that control energy homeostasis by regulating carbohydrate and lipid metabolism (2). Like other nuclear receptors, the 3 known subtypes (PPAR, PPARß/
, and PPAR) have N-terminal transactivation domains, central highly conserved DNA-binding domains, and C-terminal ligand-binding domains (LBD). Natural ligands, such as fatty acids and their derivatives, enter a pocket in the LBD (3) activating the receptor, causing it to heterodimerize with its obligate partner, the retinoid receptor. The heterodimer binds to peroxisome proliferator–responsive elements in promoter regions and recruits coregulatory molecules to modulate transcriptional activity of target genes involved in glucose metabolism and lipid homeostasis (4).
PPAR is mainly expressed in tissues such as liver, kidney, heart, and muscles where lipoprotein metabolism is important. Specific PPAR agonists, such as WY14643 (pirinixic acid), regulate genes involved in uptake of fatty acid binding proteins, ß-oxidation (acyl-CoA oxidase), and 
-oxidation (e.g., cytochrome P450[CYP]4A6) (5). PPAR is the predominant therapeutic target of the fibrates, drugs that are widely used to lower serum triglycerides and increase HDL cholesterol in patients with dyslipidaemia, atherosclerosis, coronary heart disease, and obesity. PPAR, the target for ligands such as 15-deoxy-prostaglandin J2 and thiazolidinediones, is highly expressed in adipose tissue, where it controls insulin sensitivity, adipocyte differentiation, and lipid storage. Currently, available thiazolidinediones, such as pioglitazone and rosiglitazone, are efficacious in the treatment of type II diabetes mellitus by maintaining plasma glucose and delaying the onset of long-term complications (6).
Unfortunately, pioglitazone and rosiglitazone show modest or even negative effects on blood lipid variables in patients with diabetes (7). PPAR-selective fibrates, such as fenofibrate and gemfibrozil, although efficacious in lowering triglycerides and LDL cholesterol and in raising HDL cholesterol levels in dyslipidemic patients (8), do not have sufficient activity in humans to serve as effective antidiabetic agents. Dual PPAR/ agonists that target both PPAR and PPAR may provide optimum therapeutic value for the management of the metabolic syndrome. Compounds with such properties are the focus of intense pharmaceutical research (9–11). Traditional Chinese herbal decoctions and formulations are extremely popular nutritional products consumed for their antidiabetic properties (12), largely without any understanding of their mechanisms of action. We hypothesize that small phenolic molecules present in some "antidiabetic" botanical foods may activate the PPAR-signaling system. Discovery and characterization of such putative PPAR-activating compounds would be an important first step toward their possible application in the management of the metabolic syndrome.
In this study, we used chimeric PPAR/ reporter-gene bioassays to screen for specific PPAR ligands from herbal extracts with purported antidiabetic properties. The goal was to isolate and characterize PPAR/-activating compounds from these botanicals and to compare them with currently available reference compounds.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Isolation and structural characterization of bioactive compounds. Ethanolic extracts of AM were loaded onto a medium-pressure liquid chromatography column (Buchi, 49 x 230 mm) packed with silica gel matrix (Merck, 600 g). The column was successively eluted using mixtures of hexane and acetone of increasing polarity (hexane:acetone, from 100:1 to 1:100). Ethanolic extracts of PT were also passed through a silica gel medium-pressure liquid chromatography column and eluted with the same eluting system. Further bioassay-guided separations were performed by preparative HPLC (Hypersil C18), using methanol (20–100%) and water (with 0.1% acetic acid) as the mobile phase. Structures of bioactive compounds, obtained as crystals of >95% purity, were solved through tandem MS, 1D- and 2D-NMR analyses.
Cell culture and reporter-gene bioassays. Cell culture and Treatments: Cells were obtained from American Type Culture Collection. Cervical carcinoma (HeLa) cells were grown in RPMI1640 medium. The hepatocellular carcinoma cell line HepG2 and preadipocyte 3T3-L1 cells were cultured in DMEM. All media were supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 0.1 mmol/L nonessential amino acids, and 1 mmol/L sodium pyruvate.
Plasmids pSG5PL-PPAR, pSG5PL-PPAR2, and CYP4A6-peroxisome proliferator-responsive element (PPRE)-Luc were gifts of Dr. W. Wahli, University of Lausanne, Switzerland. The pMGal4-PPAR-LBD expression plasmid was constructed by excising pSG5PL-PPAR with BstUI and BamHI and ligating proximally to the DNA binding domain of Gal4p from Saccharomyces cerevisiae. The plasmid pMGal4-PPAR-LBD was constructed by excising pSG5PL-PPAR2 with RsaI and blunt-end ligating to Hind III site of pMGal4 expression plasmid (Clontech). The upstream activating sequence (UASg)-luciferase (Luc) reporter gene was constructed by cloning 5 copies of the upstream activating sequence of Gal4p in tandem to a luciferase gene in a pGL-basic vector (14). Chimeric Gal-PPAR, by virtue of its Gal-DNA-binding domain element binds strongly to the heterologous UASg promoter of any co-transfected UASg-Luc reporter.
Reporter-gene bioassays were performed as previously described (15). Briefly, cells were seeded at 40,000 cells/well in 24-well microtiter plates and incubated for 24 h before transfection. 25 ng of the respective Gal-PPAR or full-length PPAR expression plasmids, and/or 250 ng of reporter-gene plasmid (UASg-Luc or CYP4A6-PPRE-Luc) were cotransfected into HeLa, HepG2 cells or differentiated 3T3-L1 preadipocytes with GenePORTER 2 (GTS). Transfected cells were exposed to test samples in charcoal-treated medium for 40 h. Luciferase induction responses for each treatment group were expressed as folds of vehicle-treated cells or percentages of reference drugs.
PPAR competitor binding assays.
A PPAR competitor assay (PolarScreen; Invitrogen) was applied to evaluate the binding affinity of individual isoflavones to PPAR-LBD. The assay was performed according to the manufacturer's instructions. Briefly, recombinant PPAR-LBD was added to a fluorescent PPAR ligand (fluormone PPAR-Green) to form a PPAR-LBD/fluormone complex with a high polarization index. This complex was added to individual test samples in 96-well plates, incubated at room temperature for 2 h and polarization values measured with Tecan Ultra 384 fluorescence polarization plate reader. PPAR-specific ligands in test samples displaced the fluorescent fluormone from the PPAR-LBD fluormone complex, resulting in lower polarization values. Assays were conducted in triplicate and data presented as means ± SEM. Curve fitting was performed using Prism from GraphPad Software.
Adipocyte differentiation assay. Murine fibroblast or preadipocyte 3T3-L1 cells were seeded at a density of 4 x 104 cells/well in 24-well plates and cultured to confluency for 2 d in DMEM with 10% heat inactivated fetal calf serum. Postconfluent preadipocytes were exposed to induction medium containing 10% charcoal dextran-stripped serum, insulin (5 mg/L), dexamethasone (1 µmol/L), and 3-isobutyl-1-methylxanthine (0.5 mmol/L). Induction medium was removed after 2 d; cells were washed 3 times, and exposed to increasing doses of test compounds for 8 d. Medium was replenished with appropriate ligands every 2 d. After treatment, cells were washed with PBS, fixed with 4% paraformaldehyde, and lipid droplets were stained with 0.5% oil-red O in 60% isopropanol. Stained cells were examined under phase-contrast photomicrography, and a minimum of 3 experiments were performed in duplicate.
Western blotting.
Immunoblotting of total cell lysates were performed according to standard western blotting protocol (15), using anti-PPAR monoclonal antibody (Santa Cruz Biotechnology).
Statistical analyses.
All experiments were performed at least 3 times on separate occasions. Statistical analyses were conducted using SPSS 13.0 (SPSS Inc.). One-way ANOVA was used to determine dose-dependent activities of PPAR ligands, active herbal extracts, and differential effects of isoflavones on PPAR/. For multiple comparisons with the vehicle group, Dunnett's post-hoc test was used (Fig. 2, Fig. 4D, and Fig. 5). For multiple pair-wise group comparisons, Bonferroni adjustments were applied (Fig. 1, Fig. 2B, Fig. 2C, Fig. 4B, Fig. 4C, and Supplementary Fig. 1). Repeated measurement analysis over concentration levels between AM and PT was also performed with Bonferroni correction (Fig. 2B, C). Values presented are means ± SEM and differences were considered significant at P < 0.05.
|
|
|
|
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
-specific ligand, Wy14643, dose-dependently activated the chimeric Gal-PPAR construct, with a 50% effective concentration (EC50) of 5µmol/L and a maximal activity 14-fold of the vehicle (Fig. 1A). Similarly, the PPAR-specific ligand, pioglitazone, increased the activity of the Gal-PPAR system at a maximum of 40-fold of the vehicle with an EC50 of 3 µmol/L (Fig. 1B). There was minimal cross-reaction between pioglitazone and Wy14643 with PPAR and PPAR, respectively. In addition, ligands for other steroid receptors, such as estradiol and dihydrotestosterone, did not activate chimeric receptors in these bioassays (data not shown).
Screening of "antidiabetic" herbs for PPAR activity.
Ethanolic extracts of 8 traditional Chinese herbs with purported antidiabetic properties (12) were screened for PPAR activity, using the chimeric Gal-PPAR reporter-gene bioassay (Fig. 2A). Extracts of AM and PT significantly stimulated both PPAR and PPAR. In comparison, other herbs did not exhibit any PPAR activity. At a dose of 250 mg/L, AM increased PPAR and PPAR activity up to 60% and 120% over the vehicle, respectively (Fig. 2A). Dual PPAR and PPAR activity was dose-dependent, with AM displaying higher activity than PT (Fig. 2B, C). To understand the molecular basis of the PPAR activity of AM and PT, we performed bioassay-guided fractionation of the crude extracts to isolate the PPAR-active components.
Isolation and structural characterization of PPAR-active compounds from Astragalus membranaceus and Pueraria thomsonii.
Fractionation of AM resulted in 39 fractions, of which 19 and 29 displayed strong PPAR (Fig. 3A) and PPAR (Fig. 3B) activities. Compounds in fractions 19 and 29 were present in sufficient quantities to be isolated at >95% purity. Structural characterization with mass spectrometry and NMR indicated that they contained calycosin and formononectin (Fig. 3C). Fractionation of PT with medium-pressure, liquid-chromatography silica gel resulted in 54 fractions (data not shown). PPAR-active fraction 50 was crystallized and identified as daidzein using NMR and LC-MS analysis. All 3 PPAR-active compounds characterized (calycosin, formononetin, and daidzein) were isoflavones (Fig. 4).
|
assay, biochanin A, formononetin, and genistein were the most potent activators of PPAR with EC50 of 1.3 µmol/L, <1.0 µmol/L, and 16 µmol/L, respectively, comparing favorably with WY14643 (EC50 of 5 µmol/L) (Fig. 5A, Table 1). In terms of maximal PPAR activity, biochanin A and genistein were the most efficacious, exhibiting up to 65–71% of that observed with WY14643. However, peak activity of genistein for PPAR was observed only at high doses 
30 µmol/L, unlike biochanin A, whose peak was 10-fold lower at 3 µmol/L (Fig. 5A). For PPAR, biochanin A, formononectin, and genistein were the most potent activators (EC50 of 3.7 µmol/L, 2.6 µmol/L, and 23 µmol/L vs. pioglitazone 
3 µmol/L) (Fig. 5B, Table 1). The 5 isoflavones did not differ in their maximal PPAR activities, which ranged from 20–35% of pioglitazone (Fig. 5B).
|
-ligand competitor assays.
To understand whether differences in transactivation activity were caused by differential binding affinities, we measured the ability of isoflavones to displace PPAR-LBD fluorescent-ligand complexes in a polarization competitor assay. As expected, doses of pioglitazone (at least 1 µmol/L) significantly displaced the fluormone, whereas estradiol did not (Fig. 5C). All 5 isoflavones significantly displaced fluormone at doses of at least 1 µmol/L, indicating that they bind PPAR at its LBD pocket. In this assay, calycosin and formononetin exhibited poor solubility and precipitated at doses 30 µmol/L. Excluding formononetin and calycosin, the 50% inhibitary concentration (IC50) of the other 3 compounds were in the order biochanin A < genistein < daidzein, corresponding to trends in EC50 observed with the Gal-PPAR transactivation assay (comparing Fig 5B with 5C).
Bioactivity of isoflavones using full-length PPAR assay.
To determine whether dual PPAR bioactivity of these isoflavones can be observed in a more natural context, we examined PPAR effects using full-length receptors acting on the CYP4A6-PPRE-Luc reporter vector, driven by PPRE in the promoter of the cytochrome P-450[CYP]4A gene in a hepatic (HepG2) cell line. This bioassay exhibited a lower specificity activity perhaps, reflecting the actions of endogenous ligands and effects of the heterodimeric partner retinoid-X receptor (Supplementary Fig. 1). In this full-length PPAR assay, biochanin A, formononetin and genistein exhibited the highest PPAR (EC50 of <1 µmol/L, 3.7 µmol/L, and 9.5 µmol/L, respectively) and PPAR (EC50 of <1 µmol/L, 4.3 µmol/L, 12 µmol/L) stimulating activities (Table 1). In particular, biochanin A was an order of magnitude more potent than calycosin and daidzein and comparable to values observed for WY14643 and pioglitazone for both PPAR and PPAR. Maximal activities for biochanin A reached 188 and 102% of reference drugs for PPAR and PPAR, respectively.
Effects of isoflavones on PPAR-dependent transcriptional activities and differentiation of preadipocytes.
To investigate the relevance of isoflavones on lipid metabolism, we studied their effects on the differentiation of 3T3-L1 preadipocytes, a process in which PPAR is thought to have a major role. The PPAR-driven reporter-gene CYP4A6-PPRE-Luc was transfected into differentiated preadipocytes to measure PPAR-driven responses. As expected, pioglitazone induced a significant rise in PPAR activity (4-fold vs. vehicle) (Fig. 6A). At a low dose of 3 µmol/L, only biochanin A increased activity of the CYP4A6-PPRE-Luc reporter (7.6-fold vs. vehicle). At a higher dose of 10 µmol/L, formononetin was also active (5.5-fold vs. vehicle). Western blot analysis with specific PPAR antibody indicates that these 3T3-L1 adipocytes endogenously express PPAR (Fig. 5B). These results suggest that both biochanin A and formononetin at low doses can activate endogenous PPAR in differentiated preadipocytes.
|
|
by biochanin A, genistein, and formononetin was associated with adipose differentiation, we visualized accumulation of lipid droplets with oil-red O staining. Pioglitazone, but not the vehicle or Wy14643, strongly stimulated preadipocyte differentiation, indicating the PPAR specificity of the assay (Supplemental Fig. 2). At a dose of 1 µmol/L, only biochanin A stimulated lipid droplet accumulation in preadipocytes above vehicle. Formononetin and genistein stimulated lipid accumulation at higher doses of at least 3 µmol/L and 15 µmol/L, respectively. Thus, among the isoflavones tested, biochanin A was the most potent stimulator of PPAR activity and can lead to adipocyte differentiation at 1-µmol doses.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
/ activators due to their isoflavone constituents. Furthermore closely related isoflavones exhibited significant differences in transcriptional potencies and abilities to regulate adipocyte differentiation. In the chimeric PPAR assay, formononetin and biochanin A had potencies (EC50 of <1.0 µmol/L and 1.3 µmol/L, respectively) that were comparable to those reported for recently synthesized PPAR/ agonists, such as ragaglitazar (3 µmol/L) (16), tesaglitazar (3 µmol/L) (17), and muraglitazar (0.3 µmol/L) (18). With respect to PPAR, formononetin, and biochanin A (EC50 of 2.6 µmol/L and 3.7 µmol/L, respectively) were less potent than ragaglitazar (0.093 µmol/L) (16), tesaglitazar (0.149 µmol/L) (17), and muraglitazar (0.11 µmol/L) (18). Nevertheless, these isoflavones exhibited balanced PPAR/PPAR activity ratios of 1:3 in chimeric and almost 1:1 in full-length PPAR/ assays (Table 1). This balanced-activity PPAR/PPAR profile may enhance the attractiveness of biochanin and formononetin, and foods containing them, in the management of the metabolic syndrome (1). Synthetic dual PPAR/ drugs in current development cause considerable side effects, including edema and carcinogenicity in rodent toxicity models (16–20). Isoflavones are very common botanical compounds, and epidemiological evidence suggests that their consumption may aid cancer chemoprevention (21). Our discovery of herbs and isoflavones with PPAR activity might lead to PPAR agonists with improved risk-benefit profiles. Furthermore, these isoflavones and their parent foods are available immediately for clinical evaluation.
Our data add to the increasing evidence (22–24) that isoflavones are dual PPAR/ activators. Besides being phytoestrogens, isoflavones exhibit antioxidant effects and perturb the action of DNA topoisomerase II (22). Our study indicates that closely related isoflavones have significantly different bioactivities. Biochanin A differs from genistein by only an additional methyl moiety in the phenyl B ring (Fig. 4A), but the former is several-fold more potent than the latter (Table 1). Similarly, formononetin, with an additional methyl moiety, was at least an order of magnitude more potent than its metabolite daidzein. The differences in transactivation potencies were consistently observed across several cells lines and on PPAR-regulated adipocyte differentiation, suggesting that they reflect bona fide functional differences. Differences in transactivation may be partly due to differences in binding affinity, because clear differences in the ability of isoflavone to displace bound PPAR fluormone were observed. Biochanin A and genistein displayed the strongest binding affinity, corresponding to their strong PPAR transactivity. Further molecular and structural studies are necessary to understand the mechanistic basis for these differences, whether they are related to different abilities to recruit coactivators or corepressors (25), and/or cross-activation of other steroid receptors such as estrogen receptor or RXR (26).
Isoflavones from soy (27,28) and licorice (29) exert antidiabetic and hypolipidemic effects in animal models. There is evidence that soy extracts have antilipidemic properties in humans (30), and evidence is emerging that they play a beneficial role in obesity and diabetes (31). The U.S. FDA recommends the consumption of at least 25 g of soy protein daily for cardiovascular health. Soy-based diets can result in beneficial changes to measures of glycemic control in type II diabetics (31,32). Nonetheless, isoflavones are relatively poorly absorbed, and serum concentrations seldom exceed 10 µmol/L (33,34). Our data indicate that genistein, although exerting strong maximal activity in vitro, may not have much in vivo activity due to its high EC50 values (3.0–23 µmol/L). Consumption of a soy beverage high in genistein (90 mg) results in mean genistein concentrations of only 0.6 µmol/L (35). It is not surprising that effects on dyslipidemia following administration of isoflavones based on soy products, have been mixed (28,30). Our data help explain differences following administration of preparations enriched in biochanin A compared with formononetin on lipid levels (36), based on the higher potency of the former. Although stimulation by biochanin A was weaker than that by pioglitazone, its effect on adipocyte differentiation occurred at a low dose of 1 µmol/L, a property not observed for other isoflavones. The marked differences in potency among isoflavones make it important that studies be done with foods containing precisely defined amounts of isoflavones so that nutritional effects mediated through the PPAR pathway are evident.
Our data and those of others (22–24,26,27) extend the number of potential PPAR-active compounds because isoflavones are present in many herbs and foods of botanical origins. Traditional PPAR drugs, such as pioglitazone, are potent but have serious adverse effects, such as obesity and edema. Preliminary data suggest that less potent drugs may still be efficacious while avoiding adverse effects associated with more potent ligands (37). The challenge of the future is to determine whether isoflavones with different PPAR/ potencies and their parent botanicals have any enhanced risk-benefit profiles for management of the epidemic of diabetes, dyslipidemia, and the metabolic syndrome.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Supported by NMRC/03may047 from National Medical Research Council of Singapore and NUS Academic Research Fund, Ministry of Education, Singapore. E.L.Y. is a BMRC Clinician-Scientist Investigator.
3 Supplemental Figures 1 and 2 are available with the online posting of this paper at www.nutrition.org.
5 Abbreviations used: AM, Astragalus membranaceus; EC50, 50% effective concentration; LBD, ligand-binding domain; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-responsive element; PT, Pueraria thomsonii; RXR, retinoid X receptor; UASg, upstream activating sequence of Gal4p.
Manuscript received 14 October 2005. Initial review completed 5 December 2005. Revision accepted 24 December 2005.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Eckel RH, Grundy SM, Zimmet PZ. The metabolic syndrome. Lancet. 2005;365:1415–28.[Medline]
2. Evans RM. The nuclear receptor superfamily: a rosetta stone for physiology. Mol Endocrinol. 2005;19:1429–38.
3. Issemann I, Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature. 1990;347:645–50.[Medline]
4. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995;83:841–50.[Medline]
5. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science. 2001;294:1866–70.
6. Yki-Jarvinen H. Thiazolidinediones. N Engl J Med. 2004;351:1106–18.
7. van Wijk JP, de Koning EJ, Martens EP, Rabelink TJ. Thiazolidinediones and blood lipids in type 2 diabetes. Arterioscler Thromb Vasc Biol. 2003;23:1744–9.
8. Rubins HB, Robins SJ, Collins D, Nelson DB, Elam MB, Schaefer EJ, Faas FH, Anderson JW. Diabetes, plasma insulin, and cardiovascular disease: subgroup analysis from the Department of Veterans Affairs high-density lipoprotein intervention trial (VA-HIT). Arch Intern Med. 2002;162:2597–604.
9. Guo Q, Sahoo SP, Wang PR, Milot DP, Ippolito MC, Wu MS, Baffic J, Biswas C, Hernandez M, et al. A novel peroxisome proliferator-activated receptor alpha/gamma dual agonist demonstrates favorable effects on lipid homeostasis. Endocrinology. 2004;145:1640–8.
10. Hegarty BD, Furler SM, Oakes ND, Kraegen EW, Cooney GJ. Peroxisome proliferator-activated receptor (PPAR) activation induces tissue-specific effects on fatty acid uptake and metabolism in vivo–a study using the novel PPARalpha/gamma agonist tesaglitazar. Endocrinology. 2004;145:3158–64.
11. Shi GQ, Dropinski JF, McKeever BM, Xu S, Becker JW, Berger JP, MacNaul KL, Elbrecht A, Zhou G, et al. Design and synthesis of alpha-aryloxyphenylacetic acid derivatives: a novel class of PPARalpha/gamma dual agonists with potent antihyperglycemic and lipid modulating activity. J Med Chem. 2005;48:4457–68.[Medline]
12. Kee CH. The pharmacology of Chinese herbs, 2nd ed. New York: CRC Press; 1999.
13. Yap SP, Shen P, Butler MS, Gong Y, Loy CJ, Yong EL. New estrogenic prenylflavone from Epimedium brevicornum inhibits the growth of breast cancer cells. Planta Med. 2005;71:114–9.[Medline]
14. Ghadessy FJ, Lim J, Abdullah AA, Panet-Raymond V, Choo CK, Lumbroso R, Tut TG, Gottlieb B, Pinsky L, et al. Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions. J Clin Invest. 1999;103:1517–25.[Medline]
15. Loy CJ, Sim KS, Yong EL. Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions. Proc Natl Acad Sci USA. 2003;100:4562–7.
16. Lohray BB, Lohray VB, Bajji AC, Kalchar S, Poondra RR, Padakanti S, Chakrabarti R, Vikramadithyan RK, Misra P, et al. (-)3-[4-[2-(Phenoxazin-10-yl)ethoxy]phenyl]-2-ethoxypropanoic acid [(-)DRF 2725]: a dual PPAR agonist with potent antihyperglycemic and lipid modulating activity. J Med Chem. 2001;44:2675–8.[Medline]
17. Davis T. Tesaglitazar AstraZeneca. IDrugs. 2002;5:924–6.[Medline]
18. Devasthale PV, Chen S, Jeon Y, Qu F, Shao C, Wang W, Zhang H, Cap M, Farrelly D, et al. Design and synthesis of N-[(4-methoxyphenoxy)carbonyl]-N-[[4-[2-(5- methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]methyl]glycine [Muraglitazar/BMS-298585], a novel peroxisome proliferator-activated receptor alpha/gamma dual agonist with efficacious glucose and lipid-lowering activities. J Med Chem. 2005;48:2248–50.[Medline]
19. Saad MF, Greco S, Osei K, Lewin AJ, Edwards C, Nunez M, Reinhardt RR. Ragaglitazar improves glycemic control and lipid profile in type 2 diabetic subjects: a 12-week, double-blind, placebo-controlled dose-ranging study with an open pioglitazone arm. Diabetes Care. 2004;27:1324–9.
20. Barlocco D. Muraglitazar (Bristol-Myers Squibb/Merck). Curr Opin Investig Drugs. 2005;6:427–34.[Medline]
21. Kelloff GJ, Crowell JA, Steele VE, Lubet RA, Malone WA, Boone CW, Kopelovich L, Hawk ET, Lieberman R, et al. Progress in cancer chemoprevention: development of diet-derived chemopreventive agents. J Nutr. 2000;130:467S–71S.
22. Ricketts ML, Moore DD, Banz WJ, Mezei O, Shay NF. Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review. J Nutr Biochem. 2005;16:321–30.
23. Dang ZC, Audinot V, Papapoulos SE, Boutin JA, Lowik CW. Peroxisome proliferator-activated receptor gamma (PPARgamma) as a molecular target for the soy phytoestrogen genistein. J Biol Chem. 2003;278:962–7.
24. Kim S, Shin HJ, Kim SY, Kim JH, Lee YS, Kim DH, Lee MO. Genistein enhances expression of genes involved in fatty acid catabolism through activation of PPARalpha. Mol Cell Endocrinol. 2004;220:51–8.[Medline]
25. Guan HP, Ishizuka T, Chui PC, Lehrke M, Lazar MA. Corepressors selectively control the transcriptional activity of PPARgamma in adipocytes. Genes Dev. 2005;19:453–61.
26. Dang Z, Lowik CW. The balance between concurrent activation of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis. J Bone Miner Res. 2004;19:853–61.[Medline]
27. Mezei O, Banz WJ, Steger RW, Peluso MR, Winters TA, Shay N. Soy isoflavones exert antidiabetic and hypolipidemic effects through the PPAR pathways in obese Zucker rats and murine RAW 264.7 cells. J Nutr. 2003;133:1238–43.
28. Zhan S, Ho SC. Meta-analysis of the effects of soy protein containing isoflavones on the lipid profile. Am J Clin Nutr. 2005;81:397–408.
29. Mae T, Kishida H, Nishiyama T, Tsukagawa M, Konishi E, Kuroda M, Mimaki Y, Sashida Y, Takahashi K, et al. A licorice ethanolic extract with peroxisome proliferator-activated receptor-gamma ligand-binding activity affects diabetes in KK-Ay mice, abdominal obesity in diet-induced obese C57BL mice and hypertension in spontaneously hypertensive rats. J Nutr. 2003;133:3369–77.
30. Bhathena SJ, Velasquez MT. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am J Clin Nutr. 2002;76:1191–201.
31. Li Z, Hong K, Saltsman P, DeShields S, Bellman M, Thames G, Liu Y, Wang HJ, Elashoff R, Heber D. Long-term efficacy of soy-based meal replacements vs an individualized diet plan in obese type II DM patients: relative effects on weight loss, metabolic parameters, and C-reactive protein. Eur J Clin Nutr. 2005;59:411–8.[Medline]
32. Jayagopal V, Albertazzi P, Kilpatrick ES, Howarth EM, Jennings PE, Hepburn DA, Atkin SL. Beneficial effects of soy phytoestrogen intake in postmenopausal women with type 2 diabetes. Diabetes Care. 2002;25:1709–14.
33. Bloedon LT, Jeffcoat AR, Lopaczynski W, Schell MJ, Black TM, Dix KJ, Thomas BF, Albright C, Busby MG, et al. Safety and pharmacokinetics of purified soy isoflavones: single-dose administration to postmenopausal women. Am J Clin Nutr. 2002;76:1126–37.
34. Takimoto CH, Glover K, Huang X, Hayes SA, Gallot L, Quinn M, Jovanovic BD, Shapiro A, Hernandez L, et al. Phase I pharmacokinetic and pharmacodynamic analysis of unconjugated soy isoflavones administered to individuals with cancer. Cancer Epidemiol Biomarkers Prev. 2003;12:1213–21.
35. Van Patten CL, Olivotto IA, Chambers GK, Gelmon KA, Hislop TG, Templeton E, Wattie A, Prior JC. Effect of soy phytoestrogens on hot flashes in postmenopausal women with breast cancer: a randomized, controlled clinical trial. J Clin Oncol. 2002;20:1449–55.
36. Nestel P, Cehun M, Chronopoulos A, DaSilva L, Teede H, McGrath B. A biochanin-enriched isoflavone from red clover lowers LDL cholesterol in men. Eur J Clin Nutr. 2004;58:403–8.[Medline]
37. Liu K, Black RM, Acton, 3rd JJ, Mosley R, Debenham S, Abola R, Yang M, Tschirret-Guth R, Colwell L, et al. Selective PPARgamma modulators with improved pharmacological profiles. Bioorg Med Chem Lett. 2005;15:2437–40.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L. M. Alleva, C. Cai, and I. A. Clark Using Complementary and Alternative Medicines to Target the Host Response during Severe Influenza Evid. Based Complement. Altern. Med., September 24, 2009; (2009) nep152v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Orgaard and L. Jensen The Effects of Soy Isoflavones on Obesity Experimental Biology and Medicine, September 1, 2008; 233(9): 1066 - 1080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Liu, J. Li, P. Shen, B. Husna, E. S. Tai, and E. L. Yong A Natural Polymorphism in Peroxisome Proliferator-Activated Receptor-{alpha} Hinge Region Attenuates Transcription due to Defective Release of Nuclear Receptor Corepressor from Chromatin Mol. Endocrinol., May 1, 2008; 22(5): 1078 - 1092. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
) reflect endogenous PPAR activity. (C,D) HepG2 cells transiently transfected with full-length (C) PPAR