Advertisement

Isohumulones, Bitter Acids Derived from Hops, Activate Both Peroxisome Proliferator-activated Receptor α and γ and Reduce Insulin Resistance*

Open AccessPublished:June 03, 2004DOI:https://doi.org/10.1074/jbc.M403456200
      The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARα and -γ, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARα and -γ in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARα and -γ. Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes.
      Type 2 diabetes represents a heterogeneous group of disorders characterized by increased insulin resistance. A sedentary lifestyle, fatty diet, obesity, and increased age have all been associated with the development of insulin resistance, although its molecular basis is still unknown. Thiazolidinediones were shown to improve insulin sensitivity in various animal models of diabetes (
      • Olefsky J.M.
      ) and are commonly used to treat type 2 diabetes. Thiazolidinediones directly bind to, and activate, the transcriptional factor peroxisome proliferator-activated receptor (PPAR)
      The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; IHE, isomerized hop extract; ITT, insulin tolerance test; OGTT, oral glucose tolerance test; ACO, acyl-CoA oxidase; WAT, white adipose tissue; RT, reverse transcriptase; ADRP, adipocyte differentiation-related protein; LPL, lipoprotein lipase; FAT, fatty acid translocase; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
      1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; IHE, isomerized hop extract; ITT, insulin tolerance test; OGTT, oral glucose tolerance test; ACO, acyl-CoA oxidase; WAT, white adipose tissue; RT, reverse transcriptase; ADRP, adipocyte differentiation-related protein; LPL, lipoprotein lipase; FAT, fatty acid translocase; Fmoc, N-(9-fluorenyl)methoxycarbonyl.
      γ. PPARγ is highly expressed in adipocytes where it regulates the genes responsible for growth and differentiation following activation by both natural and synthetic ligands. Activation of PPARγ was shown to not only stimulate the differentiation of adipocytes but to also induce their apoptotic death, thereby preventing adipocyte hypertrophy (
      • Yamauchi T.
      • Kamon J.
      • Waki H.
      • Murakami K.
      • Motojima K.
      • Komeda K.
      • Ide T.
      • Kubota N.
      • Terauchi Y.
      • Tobe K.
      • Miki H.
      • Tsuchida A.
      • Akanuma Y.
      • Nagai R.
      • Kimura S.
      • Kadowaki T.
      ). PPARα, another nuclear fatty acid receptor that is widely expressed in the liver, muscle, kidney, and intestine mediates the expression of genes involved in lipid metabolism. Activators of PPARα, such as fibrates, lower circulating lipid levels and are commonly used to treat hypertriglyceridemia and other dyslipidemic states. Abnormalities in fatty acid metabolism underlie the development of insulin resistance and alterations in glucose metabolism. Recent studies (
      • Ye J.M.
      • Doyle P.J.
      • Iglesias M.A.
      • Watson D.G.
      • Cooney G.J.
      • Kraegen E.W.
      ,
      • Guerre-Millo M.
      • Gervois P.
      • Raspe E.
      • Madsen L.
      • Poulain P.
      • Derudas B.
      • Herbert J.M.
      • Winegar D.A.
      • Willson T.M.
      • Fruchart J.C.
      • Berge R.K.
      • Staels B.
      ) suggested that the activation of PPARα improved the insulin resistance that was triggered by the oversupply and accumulation of lipid. It has also been reported (
      • Ye J.M.
      • Iglesias M.A.
      • Watson D.G.
      • Ellis B.
      • Wood L.
      • Jensen P.B.
      • Sorensen R.V.
      • Larsen P.J.
      • Cooney G.J.
      • Wassermann K.
      • Kraegen E.W.
      ,
      • Ljung B.
      • Bamberg K.
      • Dahllof B.
      • Kjellstedt A.
      • Oakes N.D.
      • Ostling J.
      • Svensson L.
      • Camejo G.
      ) that several novel compounds that act as co-ligands for PPARα and -γ can improve insulin sensitivity and correct diabetic dyslipidemia in obese diabetic animals.
      Attention has recently focused on the potential use of constituents in plants and other foods for the treatment of diabetic symptoms (
      • Fujita H.
      • Yamagami T.
      • Ohshima K.
      ). The treatment of type 2 diabetes with herbal plants, which was carried out generations ago in Europe, may have provided some benefit because they contained compounds that stimulated the activity of PPARα and -γ (
      • Takahashi N.
      • Kawada T.
      • Goto T.
      • Yamamoto T.
      • Taimatsu A.
      • Matsui N.
      • Kimura K.
      • Saito M.
      • Hosokawa M.
      • Miyashita K.
      • Fushiki T.
      ,
      • Swanston-Flatt S.K.
      • Day C.
      • Flatt P.R.
      • Gould B.J.
      • Bailey C.J.
      ). Hops, the female inflorescences of the hop plant (Humulus lupulus L.), are used as a preservative and flavoring in beer. Isohumulones, called iso-α acids, are the compounds that impart the bitter flavor to beer. Specifically, they are converted from humulones, i.e. α acids, derived from hops by isomerization during the brewing process. Humulone was shown to inhibit angiogenesis by suppressing cyclooxygenase-2, one of the key enzymes involved in carcinogenesis (
      • Shimamura M.
      • Hazato T.
      • Ashino H.
      • Yamamoto Y.
      • Iwasaki E.
      • Tobe H.
      • Yamamoto K.
      • Yamamoto S.
      ,
      • Yamamoto K.
      • Wang J.
      • Yamamoto S.
      • Tobe H.
      ); other than having antibacterial properties, no physiological effects were reported for isohumulones (
      • Simpson W.J.
      • Smith A.R.
      ).
      In this study, we showed that isohumulones can activate PPARα and -γ in in vitro co-transfection assays. We further demonstrated that the treatment of mildly diabetic mice and patients with isohumulones improved their insulin sensitivity.

      EXPERIMENTAL PROCEDURES

      Chemicals—Isomerized hop extract (IHE; ISOHOPCON2) was purchased from English Hop Products Co. Ltd. (Kent, UK). IHE contains isohumulones at a purity of 79%. Isohumulone, isocohumulone, and isoadhumulone were purified from IHE by centrifugal partition chromatography. Specifically, IHE was neutralized to pH 7.0 with acetic acid and subjected to centrifugal partition chromatography (25 °C, 600 rpm). Ethyl acetate and 0.1 m ammonium acetate were used as the stationary and mobile phases, respectively. Fractionated isohumulones were adjusted to pH 3.0 with HCl, after which they were extracted twice with ethyl acetate. The extracts were washed twice with saturated NaCl and the purified isohumulones dried to determine their total yields, after which they were dissolved in ethanol. Pioglitazone was purified from Actos (Takeda Chemical Industries Ltd., Osaka, Japan) by extraction with chloroform and methanol. Fenofibrate was purchased from Sigma.
      Transient Transfection Assay—An expression plasmid containing the ligand binding domain of human PPARγ fused to the GAL4 DNA binding domain was constructed as described previously (
      • Lehmann J.M.
      • Lenhard J.M.
      • Oliver B.B.
      • Ringold G.M.
      • Kliewer S.A.
      ,
      • Lehmann J.M.
      • Moore L.B.
      • Smith-Oliver T.A.
      • Wilkison W.O.
      • Willson T.M.
      • Kliewer S.A.
      ). The plasmid encoding for the full-length human PPARα fused to the GAL4 DNA binding domain was constructed in a similar manner. The PPARγ/GAL4 chimera expression plasmid and pG5luc plasmid (Promega Corp., Madison, WI) were transfected into CV-1 cells by liposomal delivery using LipofectAMINE™ (Invitrogen) according to the manufacturer's instructions. HepG2 cells were transfected with the PPARα/GAL4 chimera expression plasmid and the luciferase reporter plasmid. Isohumulones and other PPAR agonists were added to the media immediately after transfection. The cells were lysed 48 h later, and their luciferase activity and protein concentration were determined using the Luciferase Assay System (Promega Corp.) and Dc Protein Assay (Bio-Rad), respectively; luciferase activity was normalized to protein concentration. Assays were performed in triplicate.
      Animals—This study was conducted in accordance with the ethical guidelines for the animal care, handling, and termination from Kirin Pharmaceutical. Male KK-Ay mice (
      • Saha A.K.
      • Kurowski T.G.
      • Colca J.R.
      • Ruderman N.B.
      ) were purchased from Clea Japan (Tokyo, Japan). Female C57BL/6N mice were purchased from Charles River Breeding Laboratories (Tokyo, Japan). Mice were maintained under a constant 12-h light/dark cycle (light from 8:00 a.m. to 8:00 p.m.). Six-week-old male KK-Ay mice were maintained for 2 weeks either on a standard diet (
      • Reeves P.G.
      • Nielsen F.H.
      • Fahey Jr., G.C.
      ) or on a diet containing 0.18% (w/w) isohumulone, 0.18% (w/w) isocohumulone, or 0.05% (w/w) pioglitazone. Six-week-old female C57BL/6N mice were maintained on a high fat diet for 10 weeks as described previously (
      • Ikemoto S.
      • Takahashi M.
      • Tsunoda N.
      • Maruyama K.
      • Itakura H.
      • Ezaki O.
      ). Some of these animals were administered isocohumulone or isohumulone, in 0.2 m sodium carbonate buffer (pH 9.7), orally, each at doses of 10 or 100 mg/kg body weight for their final 14 days on the diet; oral glucose tolerance tests (OGTT) were then performed on these animals. Other mice similarly received either 5 or 50 mg/kg body weight of isocohumulone for 10 days, after which they were subjected to insulin tolerance testing (ITT). Plasma glucose, triglycerides, and free fatty acid levels were measured enzymatically with the glucose C II Wako, the triglyceride G test Wako, and the NEFA C Wako (Wako Pure Chemicals, Osaka, Japan), respectively.
      OGTT and ITT—For oral glucose testing, d-glucose (1g/kg body weight) was administered by stomach tube after an overnight fast. Blood samples were collected from the orbital sinus before and 15, 30, 60, and 120 min after delivery of the glucose load, under light anesthesia. For insulin tolerance testing, human insulin (Humulin R, Lilly) was injected intraperitoneally (0.75 units/kg body weight) into nonfasted animals. Blood samples were collected from the tail vein before and 15, 30, and 60 min after insulin injection, and blood glucose levels were determined using GLUTEST SENSOR (Sanwa Kagaku Kenkyusho Co., Ltd. Nagoya, Japan). Immunoreactive insulin was quantified using an insulin assay kit (Morinaga, Kanagawa, Japan). Plasma glucose level was measured as described above. The area under the glucose (1 mg/dl = 1 cm) and insulin (1 ng/ml = 1 cm) curves generated from data collected during the OGTT (60 min = 1 cm) was calculated using WinNonlin Standard Node version 3.1 software (Pharsight Corp., Mountain View, CA). The insulin resistance index (IR) was calculated by multiplying the glucose area by the insulin area, as described previously (
      • Mondon C.E.
      • Dolkas C.B.
      • Oyama J.
      ).
      RNA Preparation and Quantitative Real Time RT-PCR—Total RNA was isolated using ISOGEN (Nippon Gene, Toyama, Japan), and 5 μg of total RNA were utilized for reverse transcription using oligo(dT) primers with the ThermoScript™ RT-PCR system (Invitrogen). The reverse transcription products were used for quantitative PCR, which was carried out with the LightCycler™ PCR and Detection System using a FastStart DNA Master SYBR Green I kit (Roche Diagnostics) as described previously (
      • Cohen B.
      • Barkan D.
      • Levy Y.
      • Goldberg I.
      • Fridman E.
      • Kopolovic J.
      • Rubinstein M.
      ). Relative expression levels of the mRNA of the target genes were normalized to 36B4 (
      • Laborda J.
      ). To investigate the effect of isohumulones on PPARα and fatty acid oxidation in liver, specific primers for acyl-CoA oxidase (ACO) and fatty acid translocase/CD36 (FAT) genes were used. The primers specific for adipocyte differentiation-related protein (adipocyte-ADRP) and lipoprotein lipase (LPL), targets for PPARγ and involved in lipid uptake and storage, were also used for the analysis. The following sense and antisense primers were used (GenBank™ accession numbers are in parentheses) as follows: 36B4 (X15267), nucleotides 740-759 and 907-929; adipocyte-ADRP (NM024406), nucleotides 218-236 and 364-385; LPL (NM008509), nucleotides 37-58 and 242-265; ACO (AF006688), nucleotides 1392-1415 and 1607-1628; FAT (L23108), nucleotides 331-352 and 509-531.
      Acyl-CoA Oxidase Activity—Activity of ACO was determined as described previously (
      • Kushiro M.
      • Masaoka T.
      • Hageshita S.
      • Takahashi Y.
      • Ide T.
      • Sugano M.
      ). Briefly, 6-week-old female C57BL/6N mice fed a standard diet were orally administered 100 mg/kg body weight of isohumulone or isocohumulone for 6 days. Their livers were then isolated and homogenized in a solution containing 0.25 m sucrose, 1 mm EDTA, and 3 mm Tris-Cl (pH 7.2). The homogenates were centrifuged at 500 × g for 10 min, and their supernatants were used as an enzyme source, and peroxisomal palmitoyl-CoA oxidation rates were analyzed.
      Histological Analysis of Adipose Tissue and Determination of Adipocyte Size—C57BL/6N mice that were fed a high fat diet for 10 weeks were orally administered isocohumulone or isohumulone at a dose of 100 mg/kg weight, or pioglitazone at a dose of 10 mg/kg weight, for 10 days after which their subcutaneous white adipose tissue was removed and fixed in 4% paraformaldehyde in phosphate-buffered saline. Fixed specimens were dehydrated, embedded in tissue-freezing medium (Tissue Tek OTC compound; Sakura Finetechnical Co., Ltd., Tokyo, Japan), and frozen in liquid nitrogen. Sections (14 μm) were cut and stained with hematoxylin and eosin. Adipocyte size was determined using Win-ROOF software (Mitani Co. Ltd., Fukui, Japan). More than 200 cells/mouse in each group were analyzed as described previously (
      • Kubota N.
      • Terauchi Y.
      • Miki H.
      • Tamemoto H.
      • Yamauchi T.
      • Komeda K.
      • Satoh S.
      • Nakano R.
      • Ishii C.
      • Sugiyama T.
      • Eto K.
      • Tsubamoto Y.
      • Okuno A.
      • Murakami K.
      • Sekihara H.
      • Hasegawa G.
      • Naito M.
      • Toyoshima Y.
      • Tanaka S.
      • Shiota K.
      • Kitamura T.
      • Fujita T.
      • Ezaki O.
      • Aizawa S.
      • Kadowaki T.
      ). Other sections were stained using the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique (Mebstain Apoptosis Kit Direct, Medical and Biological Laboratories Co. Ltd., Nagoya, Japan) to detect apoptotic nuclei.
      Double-blind, Placebo-controlled Pilot Study in Type 2 Diabetics—This study was approved by the ethics committee of Kirin Brewery Co., Ltd., and complied with the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all subjects. Twenty volunteers with mild type 2 diabetes (both males and females) were included in this study. Ten men and ten women were randomized into one of two groups receiving either placebo or a capsule containing 100 mg of IHE. The subjects were between 45 and 65 years of age (mean = 53 years) and had fasting blood glucose and hemoglobin A1c levels between 110 and 140 (mg/dl) and 6.4 and 8.0 (%), respectively. IHE, containing isohumulones at a purity of 79%, was used in this study. The ability of IHE to transactivate PPARα and -γ and to improve glucose tolerance in KK-Ay mice was identical to that seen with isohumulones isolated from the extract (data not shown). The subjects were randomized to one of two groups that received either a placebo or a capsule containing 100 mg of IHE (equivalent to about 80 mg of isohumulones) twice a day for 12 weeks. Laboratory tests were performed every 4 weeks for 12 weeks.
      Statistical Analysis—Results are expressed as the means ± S.D. The data in the animal experiments were analyzed by nonrepeated measures analysis of variance followed by a Dunnett's test. Significance was assumed if the p value was <0.05.

      RESULTS

      Isohumulones Activate Both PPARα and PPARγ—Fig. 1 shows the chemical structure of three major homologs of isohumulones, isohumulone, isocohumulone, and isoadhumulone. The effects of isohumulones on PPARα and -γ were evaluated in transient co-transfection assays using mammalian cell lines. Because mammalian cell lines contain endogenous nuclear receptors that can complicate the interpretation of the results, chimera systems were used in which the ligand binding domain of PPARα or PPARγ was fused to the DNA binding domain of the yeast transcription factor GAL4 (
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ). To evaluate the ability for transactivation of PPARγ, an expression vector encoding chimeric PPARγ and GAL4 was transfected into CV-1 cells with a reporter plasmid containing the reporter luciferase gene with five GAL4 binding sites in the promoter region. In the case for PPARα, a chimeric plasmid encoding the full-length of PPARα protein fused to the DNA binding domain of GAL4 was used, because the chimeric construct containing PPARα ligand binding and GAL4 DNA binding domains did not provide enough luciferase activity in the presence of a PPARα agonist, fenofibrate (data not shown). Furthermore, human hepatoma cells, HepG2, instead of CV-1 were used for the assay to obtain a higher sensitivity.
      Figure thumbnail gr1
      Fig. 1Structure of isohumulone. Isohumulone (R: -CH3CH-(CH3)2; 2-(3-methylbutanoyl)-5-(3-methyl-2-butenyl)-3,4-dihydroxy-4-(4-methyl-3-pentenoyl)-2-cyclopentenone), isocohumulone (R: -CH(CH3)2; 2-(2-methylpropanoyl)-5-(3-methyl-2-butenyl)-3,4-dihydroxy-4-(4-methyl-3-pentenoyl)-2-cyclopentenone), and isoadhumulone (R: -CH(CH3)C2H5; 2-(2-methylbutanoyl)-5-(3-methyl-2-butenyl)-3,4-dihydroxy-4-(4-methyl-3-pentenoyl)-2-cyclopentenone).
      A dose-dependent increase in luciferase activity was observed following the addition of isohumulones to cells transfected with the PPARγ/GAL4 chimera construct. The addition of either 10 μm isohumulone, isocohumulone, or isoadhumulone induced a 3.8-, 3.5-, and 2.8-fold increase in luciferase activity, respectively, compared with the vehicle control (Fig. 2A). These activities were almost the same as those seen when 1 μm pioglitazone, a specific agonist of PPARγ, was added to the cells. Isohumulone and isocohumulone also activated the PPARα/GAL4 chimera construct in a dose-dependent manner; 10 μm isohumulone and isocohumulone increased luciferase activity by about 3.2- and 1.9-fold, respectively, compared with the vehicle control (Fig. 2B). Isoadhumulone had no effect on the PPARα/GAL4 chimera construct, suggesting that the side chain of isohumulone may be involved in the activation of the PPARα receptor. The activity of isohumulone at 30 μm was almost the same as that of 3 μm fenofibrate, a PPARα-selective agonist.
      Figure thumbnail gr2
      Fig. 2Isohumulone-induced PPAR transactivation.A, CV-1 cells were co-transfected with a luciferase reporter plasmid, pG5 luc, containing five copies of GAL4 upstream activating sequence in the promoter region and an expression vector for the human PPARγ ligand binding domain fused to the GAL4 DNA binding domain. Results are the relative luciferase expression levels normalized with the protein concentration of the cell lysates. B, HepG2 cells were co-transfected with pG5 luciferase, and an expression vector for the human PPARα coding region fused to the GAL4 DNA binding domain. Isohumulone (IH), isocohumulone (IcH), and isoadhumulone (IaH) at the indicated concentrations were added to the transfected cells. Pioglitazone (Pio) and fenofibrate (Feno) were used as positive controls for PPARγ and PPARα transactivation, respectively.
      Isohumulones Prevent the Development of Diabetes in KK-Ay Mice—Treatment of KK-Ay mice with isohumulones for 2 weeks significantly lowered their plasma triglyceride (62.6% for isohumulone and 76.4% for isocohumulone, respectively) and free fatty acid levels (73.1% for isohumulone and 84.8% for isocohumulone, respectively). These reductions were comparable with those seen in 0.05% pioglitazone-treated mice (60.5 and 69.9%, respectively) (Fig. 3, A and B). The nonfasting plasma glucose levels of isohumulones-treated mice were also reduced to 65.3% of controls for isohumulone and 87.1% of controls for isocohumulone, although they were higher than those seen in animals fed pioglitazone (35.1%) (Fig. 3C). Most interesting, there was a significant difference in body weight gain between these groups despite their equivalent caloric intake. Thus, pioglitazone induced a 10% increase in body weight, whereas isohumulones did not induce an increase in body weight compared with the control group (Fig. 3D).
      Figure thumbnail gr3
      Fig. 3Isohumulone and isocohumulone prevented the development of diabetes in KK-Ay mice. Male KK-Ay mice were fed either a standard diet or a diet containing 0.18% isohumulone (IH), 0.18% isocohumulone (IcH), or 0.05% pioglitazone (Pio) for 2 weeks. Fasting blood samples were collected, and plasma triglyceride and free fatty acid levels were measured (A and B). Nonfasting plasma glucose levels were also determined (C), and body weights were measured weekly (D). Data are the means ± S.D. of six mice per group. *, p < 0.05; **, p < 0.01 versus control (Cont).
      Isohumulones Reduced Insulin Resistance in High Fat Dietfed C57BL/6N Mice—Female C57BL/6N mice were fed a high fat diet for 12 weeks. On their last 14 days on the diet, they were orally administered isocohumulone. OGTTs and ITTs were performed on their 10th and 14th day of isocohumulone treatment, respectively. After glucose loading, plasma glucose levels in the mice treated with isocohumulone at 10 and 100 mg/kg/day were significantly reduced compared with mice treated with vehicle at all time points except for the mouse treated with 100 mg/kg of isocohumulone at 120 min (Fig. 4A). Fasting plasma insulin levels were significantly reduced in animals treated with 100 mg/kg isocohumulone (2396 ± 520 and 1605 ± 570 (pg/ml) for vehicle-treated and 100 mg isocohumulone-treated mice, respectively). Plasma insulin levels during the OGTT were also reduced in the isocohumulone-treated animals (Fig. 4B). The insulin resistance indices of mice treated with isocohumulone (1094.4 ± 259.2 and 1034.0 ± 259.2 for 10 mg of isocohumulone-treated and 100 mg of isocohumulone-treated mice, respectively) were significantly (p < 0.01 using the Dunnett's test) lower than that seen in the control group (2217.3 ± 792.9), indicating that isocohumulone improved insulin sensitivity in mice fed a high fat diet. Improvement in insulin sensitivity was also observed by the treatment with isohumulone; significant decrease in IR was observed in the group receiving 100 mg/kg of isohumulone (1240.2 ± 259.5, p < 0.01 using the Dunnett's test, data not shown). Results of the ITT showed a greater glucose lowering effect in mice treated with isocohumulone for 10 days than in vehicle-treated animals (Fig. 4C). Plasma glucose levels in mice treated with 5 or 50 mg/kg of isocohumulone were significantly reduced to 76.8 and 69.3% of controls after 60 min and to 82.8 and 69.3% of controls after 120 min.
      Figure thumbnail gr4
      Fig. 4Isocohumulone ameliorated insulin resistance in high fat diet-fed C57BL/6N mice. Female C57BL/6N mice were fed a high fat diet for 10 weeks. For glucose tolerance testing, isocohumulone (IcH) was orally administered to mice at a dose of 10 or 100 mg/kg body weight for 14 days. The mice were then subjected to an OGTT. Control (Ctrl) mice received buffer alone. Plasma glucose (A) and insulin (B) concentrations were plotted on the graph (mean ± S.D., n = 5-6). For insulin tolerance testing, high fat diet-fed mice were administered isocohumulone at a dose of 5 or 50 mg/kg body weight for 10 days. Blood glucose concentrations during the ITT were plotted on the graph (mean ± S.D., n = 6) (C). *, p < 0.05; **, p < 0.01 for vehicle versus treatment with isocohumulone at each indicated time.
      Isohumulones Up-regulated the Expression of Genes Involved in Fatty Acid Oxidation in the Liver—Quantitative real time RT-PCR analysis of the mRNAs for ACO and fatty acid translocase/CD36 (FAT) genes in the liver of KK-Ay mice treated with isohumulone, isocohumulone, or pioglitazone were performed (Fig. 5A). ACO and FAT, whose expressions are regulated by PPARα, are involved in peroxisomal β-oxidation of fatty acids and in the uptake of long chain fatty acids through the cell membrane, respectively (
      • Memon R.A.
      • Tecott L.H.
      • Nonogaki K.
      • Beigneux A.
      • Moser A.H.
      • Grunfeld C.
      • Feingold K.R.
      ,
      • Motojima K.
      • Passilly P.
      • Peters J.M.
      • Gonzalez F.J.
      • Latruffe N.
      ). Treatment of mice with isohumulone, isocohumulone, or pioglitazone increased ACO and its mRNA levels by 1.6-, 1.7-, and 2.7-fold, and FAT mRNA levels by 2.9-, 3.3-, and 2.8-fold, compared with the control mice, respectively. The significant increase in ACO and FAT mRNA levels in the pioglitazone-treated mice was likely due to activation of PPARα by pioglitazone because induction of PPARα/GAL4 chimera transactivation with pioglitazone was observed in our reporter assay (date not shown). An increase in the activity of ACO was observed in the liver of C57BL/6N mice treated with isohumulone or isocohumulone (Fig. 5B).
      Figure thumbnail gr5
      Fig. 5Effects of isohumulone and isocohumulone on liver and white adipose tissue.A and C, total RNA was isolated from liver and subcutaneous adipose tissue of KK-Ay mice fed diets containing 0.18% isohumulone (IH), 0.18% isocohumulone (IcH), or 0.05% pioglitazone (pio). Quantitative RT-PCR was performed to measure the mRNA levels of liver ACO and FAT (A), and adipocyte-ADRP and LPL in white adipose tissue (WAT) (C). LPL, lipoprotein lipase. Results are presented as relative expression levels normalized to the expression in the control (Cont) group (mean ± S.D., n = 6). B, ACO activity in the liver of C57BL/6N mice treated with isohumulone or isocohumulone at 100 mg/kg weight for 6 days. Data are presented as the amount of hydrogen peroxide produced by oxidation of palmitoyl-CoA in the homogenates. *, p < 0.05; **, p < 0.01 versus control (Ctrl). D, histological analysis and cell-size distribution of subcutaneous WAT in C57BL/6N mice. High fat diet-fed mice were orally administered vehicle (Cont), isohumulone, isocohumulone (100 mg/kg weight), or pioglitazone (Pio) (10 mg/kg weight) for 2 weeks. E, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining of representative sections of white adipose tissue from D. Bar indicates 100 μm.
      Isohumulones Reduced Adipocyte Hypertrophy in White Adipose Tissue—Isohumulone treatment unexpectedly resulted in only a fairly small increase in the expression of PPARγ-regulated adipocyte-ADRP and LPL genes, which are involved in lipid uptake and storage in the epididymal white adipose tissue (WAT) (
      • Tontonoz P.
      • Hu E.
      • Spiegelman B.M.
      ) of KK-Ay mice; only the LPL mRNA level in the mice treated with isocohumulone significantly increased by 1.2-fold, although treatment with pioglitazone did increase the adipocyte-ADRP and LPL mRNA levels by 2- and 1.7-fold, respectively (Fig. 5C). Histological analysis of subcutaneous WAT of the high fat diet-fed C57BL/6N mice treated with isohumulone, isocohumulone, or pioglitazone for 10 days revealed an increase in the number of small adipocytes by treatment with all three of these compounds compared with vehicle-treated mice (Fig. 5D). We also examined whether isohumulones induced apoptosis in adipocytes. Our results indicated that the treatment of high fat diet-fed mice with isohumulones for 10 days induced the apoptosis of hypertrophic adipocytes, which was also observed in the mice treated with pioglitazone (Fig. 5E).
      The Effects of Oral Isohumulones in Type 2 Diabetics in a Placebo-controlled Pilot Study—The ability of IHE to transactivate PPARα and -γ and to improve glucose tolerance in KK-Ay mice was identical to that seen with isohumulones isolated from the extract (data not shown). Supplementation of 10 mild diabetic patients with IHE over 8 weeks resulted in a significant reduction in their blood glucose and hemoglobin A1c levels by 10.1 and 6.4%, respectively; most interesting, a weak but significant reduction in blood glucose levels (7.3%) was also observed in the placebo control group. Furthermore, significant reductions in systolic blood pressure and blood levels of glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and γGTP were observed in the IHE-treated group (7.2, 33.4, 21.3, and 25.6% reduction, respectively) (Table I).
      Table IResults of the placebo-controlled pilot study of the effects of hop extract in type 2 diabetic patients
      Hop extractPlacebo
      0 week8 weeks0 week8 weeks
      Blood glucose (mg/dl)127.1 ± 10.9114.3 ± 10.4
      p < 0.01 versus 0 week of each group.
      130.5 ± 12.0122.3 ± 8.37
      p < 0.05.
      HbA1c (%)7.14 ± 0.366.68 ± 0.68
      p < 0.01 versus 0 week of each group.
      7.00 ± 0.386.76 ± 0.65
      Systolic blood pressure (mm Hg)137.1 ± 13.6128.6 ± 15.5
      p < 0.01 versus 0 week of each group.
      129.0 ± 16.4127.5 ± 15.3
      GPT
      GPT indicates glutamic pyruvic transaminase.
      (IU/liter)
      40.8 ± 2627.2 ± 11.0
      p < 0.01 versus 0 week of each group.
      25.4 ± 18.024.8 ± 23
      GOT
      GOT indicates glutamic oxaloacetate transaminase.
      (IU/liter)
      28.6 ± 1322.5 ± 6.30
      p < 0.05.
      25.7 ± 10.027.0 ± 16
      γGTP (IU/liter)48.1 ± 4135.8 ± 26.0
      p < 0.05.
      75.3 ± 91.078.3 ± 110
      a p < 0.01 versus 0 week of each group.
      b p < 0.05.
      c GPT indicates glutamic pyruvic transaminase.
      d GOT indicates glutamic oxaloacetate transaminase.

      DISCUSSION

      Metabolic syndrome, which is particularly relevant to insulin resistance, is characterized by glucose intolerance, hyperinsulinemia, dyslipidemia, and hypertension and is frequently associated with visceral obesity. The clustering of these multiple cardiovascular risk factors increases the risk of developing atherosclerotic vascular disease. Pharmacological treatment of this syndrome aims to reduce insulin resistance and other risk factors by modulating PPARs, nuclear hormone receptors that play a key role in regulating energy metabolism. Fibrate drugs, which act as ligands for PPARα and thiazolidinedione drugs, which are ligands for PPARγ, are often used to treat hyperlipidemia and hyperglycemia, respectively. The combination therapy of these medications is an attractive option for the treatment of obese type 2 diabetics (
      • Shimamura M.
      • Hazato T.
      • Ashino H.
      • Yamamoto Y.
      • Iwasaki E.
      • Tobe H.
      • Yamamoto K.
      • Yamamoto S.
      ,
      • Yamamoto K.
      • Wang J.
      • Yamamoto S.
      • Tobe H.
      ). Compounds that have dual agonistic activity on both of these receptors have been shown to improve insulin sensitivity and dyslipidemia in obese diabetic animals (
      • Ljung B.
      • Bamberg K.
      • Dahllof B.
      • Kjellstedt A.
      • Oakes N.D.
      • Ostling J.
      • Svensson L.
      • Camejo G.
      ,
      • Murakami K.
      • Tobe K.
      • Ide T.
      • Mochizuki T.
      • Ohashi M.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ). In the present study, we showed that isohumulones, the bitter compounds present in beer, activated PPARs α and γ ameliorated insulin resistance and dyslipidemia in diabetic animals and reduced hyperglycemia in patients with type 2 diabetes.
      Our experiments using an in vitro reporter assay indicated that isohumulones activated transcription that was mediated by PPARα and PPARγ, suggesting that isohumulones may modulate reactions that are regulated by these transcriptional activators. When KK-Ay mice, a model of noninsulin-dependent diabetes, were treated with isohumulone and isocohumulone, their hyperglycemia and hyperlipidemia improved, as they also did following treatment with pioglitazone, a thiazolidinedione drug. However, treatment did not cause significant body weight gain nor marked increases in mRNA levels of PPARγ-regulated adipocyte-ADRP and LPL genes, which are involved in lipid uptake and storage in adipose tissue, in contrast to pioglitazone. It has been suggested that Fmoc-l-leucine, a potent insulin-sensitizing compound, acts as a selective PPARγ modulator and improves insulin resistance in diabetic mice despite only marginally inducing adipocyte-ADRP and LPL gene expression in adipose tissue (
      • Rocchi S.
      • Picard F.
      • Vamecq J.
      • Gelman L.
      • Potier N.
      • Zeyer D.
      • Dubuquoy L.
      • Bac P.
      • Champy M.F.
      • Plunket K.D.
      • Leesnitzer L.M.
      • Blanchard S.G.
      • Desreumaux P.
      • Moras D.
      • Renaud J.P.
      • Auwerx J.
      ). It should be noted that treatment with this compound also had no effect on body weight. A low affinity PPARγ antagonist, i.e. LG100,641, has also been shown to improve insulin sensitivity in 3T3-L1 adipocytes in vitro without markedly up-regulating PPARγ-mediated gene expression and adipocyte differentiation (
      • Mukherjee R.
      • Hoener P.A.
      • Jow L.
      • Bilakovics J.
      • Klausing K.
      • Mais D.E.
      • Faulkner A.
      • Croston G.E.
      • Paterniti Jr., J.R.
      ). Thus, it is likely that an increase in body weight and up-regulation of PPARγ-mediated gene expression in adipose tissue are not always observed in rodents treated with PPARγ agonists.
      Mice fed a high fat diet develop hyperglycemia and obesity and are used as a model of noninsulin-dependent diabetes mellitus (
      • Ikemoto S.
      • Takahashi M.
      • Tsunoda N.
      • Maruyama K.
      • Itakura H.
      • Ezaki O.
      ). Insulin resistance and glucose intolerance in high fat diet-fed obese mice improved after short term oral administration of isohumulone and isocohumulone. Their adipocyte hypertrophy also responded to treatment, with an increase seen in the number of apoptotic adipocytes in the isohumulone-treated groups; similar results were found with pioglitazone. It has been suggested that treatment of obese diabetic mice with thiazolidinediones might stimulate adipocyte differentiation and apoptosis and thereby prevent adipocyte hypertrophy. In fact, such treatment was shown to be associated with reduced insulin resistance that may have been due to reductions in free fatty acid and tumor necrosis factor-α levels, and up-regulation of adiponectin (
      • Yamauchi T.
      • Kamon J.
      • Waki H.
      • Murakami K.
      • Motojima K.
      • Komeda K.
      • Ide T.
      • Kubota N.
      • Terauchi Y.
      • Tobe K.
      • Miki H.
      • Tsuchida A.
      • Akanuma Y.
      • Nagai R.
      • Kimura S.
      • Kadowaki T.
      ,
      • Yamauchi T.
      • Waki H.
      • Kamon J.
      • Murakami K.
      • Motojima K.
      • Komeda K.
      • Miki H.
      • Kubota N.
      • Terauchi Y.
      • Tsuchida A.
      • Tsuboyama-Kasaoka N.
      • Yamauchi N.
      • Ide T.
      • Hori W.
      • Kato S.
      • Fukayama M.
      • Akanuma Y.
      • Ezaki O.
      • Itai A.
      • Nagai R.
      • Kimura S.
      • Tobe K.
      • Kagechika H.
      • Shudo K.
      • Kadowaki T.
      ). Taken together, these results suggest that isohumulones improve insulin sensitivity by acting on adipose tissues.
      Our in vitro reporter assay results showed that isohumulone and isocohumulone activated PPARα-mediated gene expression. It was therefore likely that the increase in the mRNA levels of ACO and fatty acid translocase/CD36 genes, which are involved in fatty acid oxidation, in the liver of KK-Ay mice treated with isohumulones was due to the activation of PPARα. The mRNA for medium chain acyl-CoA dehydrogenase, an enzyme involved in fatty acid β-oxidation, was also increased in the liver by this treatment (data not shown). An increase in the enzymatic activity of ACO was observed in the liver of C57BL/6N mice treated with isohumulones. These results may indicate that isohumulones facilitate lipid metabolism in the liver. It has been reported that fenofibrate reduced plasma insulin and glucose levels in high fat diet-induced C57BL/6 mice and obese Zucker and OLETF rats, whereas it prevented weight gain and expansion of adipose tissue mass (
      • Guerre-Millo M.
      • Gervois P.
      • Raspe E.
      • Madsen L.
      • Poulain P.
      • Derudas B.
      • Herbert J.M.
      • Winegar D.A.
      • Willson T.M.
      • Fruchart J.C.
      • Berge R.K.
      • Staels B.
      ,
      • Lee H.J.
      • Choi S.S.
      • Park M.K.
      • An Y.J.
      • Seo S.Y.
      • Kim M.C.
      • Hong S.H.
      • Hwang T.H.
      • Kang D.Y.
      • Garber A.J.
      • Kim D.K.
      ). The activation of hepatic β-oxidation in isohumulone-treated obese C57BL/6N mice may have been the mechanism by which adipocyte hypertrophy was reduced and at least partly responsible for the amelioration of insulin resistance. However, it should be noted that no improvement in hyperglycemia and hyperlipidemia was observed in KK-Ay mice that were treated with fenofibrate, suggesting that activation of PPARα alone was not sufficient to ameliorate insulin resistance in this model (data not shown). Anti-diabetic effects of dual PPARα and PPARγ agonists have been reported over the past several years. Ragaglitazar, a PPARα and -γ agonist, was shown to reduce plasma triglyceride and glucose levels in high fat diet-fed animals in the absence of weight gain (
      • Ye J.M.
      • Iglesias M.A.
      • Watson D.G.
      • Ellis B.
      • Wood L.
      • Jensen P.B.
      • Sorensen R.V.
      • Larsen P.J.
      • Cooney G.J.
      • Wassermann K.
      • Kraegen E.W.
      ); similar results were observed in Zucker fa/fa rats (
      • Chakrabarti R.
      • Vikramadithyan R.K.
      • Misra P.
      • Hiriyan J.
      • Raichur S.
      • Damarla R.K.
      • Gershome C.
      • Suresh J.
      • Rajagopalan R.
      ). Another PPARα/γ co-ligand, KRP-297, was shown to reduce plasma glucose and insulin levels in ob/ob and db/db mice (
      • Kubota N.
      • Terauchi Y.
      • Miki H.
      • Tamemoto H.
      • Yamauchi T.
      • Komeda K.
      • Satoh S.
      • Nakano R.
      • Ishii C.
      • Sugiyama T.
      • Eto K.
      • Tsubamoto Y.
      • Okuno A.
      • Murakami K.
      • Sekihara H.
      • Hasegawa G.
      • Naito M.
      • Toyoshima Y.
      • Tanaka S.
      • Shiota K.
      • Kitamura T.
      • Fujita T.
      • Ezaki O.
      • Aizawa S.
      • Kadowaki T.
      ). The co-activation of PPARα and -γ by isohumulones, leading to the activation of fatty acid β-oxidation in the liver and the normalization of adipocyte hypertrophy, may be responsible for the amelioration of insulin resistance in diabetic mice. Recently, it was reported (
      • Wang Y.X.
      • Lee C.H.
      • Tiep S.
      • Yu R.T.
      • Ham J.
      • Kang H.
      • Evans R.M.
      ,
      • Dressel U.
      • Allen T.L.
      • Pippal J.B.
      • Rohde P.R.
      • Lau P.
      • Muscat G.E.
      ) that the activation of PPARδ increased fatty acid oxidation and resulted in the prevention of obesity. It should be noted that the activation of PPARδ with isohumulones was not observed in in vitro reporter assays (data not shown).
      Natural constituents in plants that are present in folk remedies as well as traditional foods have been reported to be effective for the treatment and prevention of lifestyle-related diseases (
      • Fujita H.
      • Yamagami T.
      • Ohshima K.
      ,
      • Pereira M.A.
      • Jacobs Jr., D.R.
      • Pins J.J.
      • Raatz S.K.
      • Gross M.D.
      • Slavin J.L.
      • Seaquist E.R.
      ). A recent study demonstrated that the plant sterol guggulusterone, derived from Commiphora mukul (guggulu in Sanskrit), which has been used in Ayurvedic medicine, acts as an antagonist for the nuclear hormone receptor, farnesoid X receptor, and lowers low density lipoprotein levels in rodents and humans (
      • Singh V.
      • Kaul S.
      • Chander R.
      • Kapoor N.K.
      ). Hops, which have been used as an herbal medicine for many years, were shown to contain several compounds that have significant biological effects, such as 8-prenylnaringenin, which has estrogenic activity (
      • Milligan S.
      • Kalita J.
      • Pocock V.
      • Heyerick A.
      • De Cooman L.
      • Rong H.
      • De Keukeleire D.
      ), and xanthohumol and hexahydro-colupulone, which were shown to inhibit the growth of cancers (
      • Gerhauser C.
      • Alt A.
      • Heiss E.
      • Gamal-Eldeen A.
      • Klimo K.
      • Knauft J.
      • Neumann I.
      • Scherf H.R.
      • Frank N.
      • Bartsch H.
      • Becker H.
      ). Humulone was also shown to have anti-tumor effects (
      • Shimamura M.
      • Hazato T.
      • Ashino H.
      • Yamamoto Y.
      • Iwasaki E.
      • Tobe H.
      • Yamamoto K.
      • Yamamoto S.
      ,
      • Yamamoto K.
      • Wang J.
      • Yamamoto S.
      • Tobe H.
      ,
      • Yasukawa K.
      • Takeuchi M.
      • Takido M.
      ). Isohumulones, which are isomerized humulones, are present in beer. To date, most studies of the therapeutic effects of isohumulones have focused on their ability to inhibit bacterial growth (
      • Simpson W.J.
      • Smith A.R.
      ). In our study, isohumulone and isocohumulone were shown to activate PPARα and -γ, whereas isoadhumulone only activated PPARγ; the reason for the differing effects of these compounds is unclear. Further studies are required to determine the molecular basis of the interaction between isohumulones and PPARs.
      In conclusion, our results suggest that isohumulones, the bitter compounds derived from hops in beer, activate both PPARα and -γ and improve insulin sensitivity and lipid metabolism. The simultaneous activation of PPARα and -γ with isohumulones may be an effective approach for the treatment of metabolic syndrome in which glucose and lipid metabolism are both impaired. Results of a preliminary clinical study indicated that isohumulones improved insulin sensitivity in type 2 diabetic patients. Most interesting, in this latter study, patients also showed a significant reduction in systolic blood pressure and in the levels of serum markers of liver disorder. Thus, isohumulones in beer are worth investigating further as therapeutic agents for the treatment of metabolic syndrome associated with insulin resistance.

      Acknowledgments

      We thank H. Watanabe and K. Akatsuka for their hands-on assistance with these experiments, and Dr. K. Kubo for calculating the IR index.

      References

        • Olefsky J.M.
        J. Clin. Investig. 2000; 106: 467-472
        • Yamauchi T.
        • Kamon J.
        • Waki H.
        • Murakami K.
        • Motojima K.
        • Komeda K.
        • Ide T.
        • Kubota N.
        • Terauchi Y.
        • Tobe K.
        • Miki H.
        • Tsuchida A.
        • Akanuma Y.
        • Nagai R.
        • Kimura S.
        • Kadowaki T.
        J. Biol. Chem. 2001; 276: 41245-41254
        • Ye J.M.
        • Doyle P.J.
        • Iglesias M.A.
        • Watson D.G.
        • Cooney G.J.
        • Kraegen E.W.
        Diabetes. 2001; 50: 411-417
        • Guerre-Millo M.
        • Gervois P.
        • Raspe E.
        • Madsen L.
        • Poulain P.
        • Derudas B.
        • Herbert J.M.
        • Winegar D.A.
        • Willson T.M.
        • Fruchart J.C.
        • Berge R.K.
        • Staels B.
        J. Biol. Chem. 2000; 275: 16638-16642
        • Ye J.M.
        • Iglesias M.A.
        • Watson D.G.
        • Ellis B.
        • Wood L.
        • Jensen P.B.
        • Sorensen R.V.
        • Larsen P.J.
        • Cooney G.J.
        • Wassermann K.
        • Kraegen E.W.
        Am. J. Physiol. 2003; 284: E531-E540
        • Ljung B.
        • Bamberg K.
        • Dahllof B.
        • Kjellstedt A.
        • Oakes N.D.
        • Ostling J.
        • Svensson L.
        • Camejo G.
        J. Lipid Res. 2002; 43: 1855-1863
        • Fujita H.
        • Yamagami T.
        • Ohshima K.
        J. Nutr. 2001; 131: 2105-2108
        • Takahashi N.
        • Kawada T.
        • Goto T.
        • Yamamoto T.
        • Taimatsu A.
        • Matsui N.
        • Kimura K.
        • Saito M.
        • Hosokawa M.
        • Miyashita K.
        • Fushiki T.
        FEBS Lett. 2002; 514: 315-322
        • Swanston-Flatt S.K.
        • Day C.
        • Flatt P.R.
        • Gould B.J.
        • Bailey C.J.
        Diabetes Res. 1989; 10: 69-73
        • Shimamura M.
        • Hazato T.
        • Ashino H.
        • Yamamoto Y.
        • Iwasaki E.
        • Tobe H.
        • Yamamoto K.
        • Yamamoto S.
        Biochem. Biophys. Res. Commun. 2001; 289: 220-224
        • Yamamoto K.
        • Wang J.
        • Yamamoto S.
        • Tobe H.
        FEBS Lett. 2000; 465: 103-106
        • Simpson W.J.
        • Smith A.R.
        J. Appl. Bacteriol. 1992; 72: 327-334
        • Lehmann J.M.
        • Lenhard J.M.
        • Oliver B.B.
        • Ringold G.M.
        • Kliewer S.A.
        J. Biol. Chem. 1997; 272: 3406-3410
        • Lehmann J.M.
        • Moore L.B.
        • Smith-Oliver T.A.
        • Wilkison W.O.
        • Willson T.M.
        • Kliewer S.A.
        J. Biol. Chem. 1995; 270: 12953-12956
        • Saha A.K.
        • Kurowski T.G.
        • Colca J.R.
        • Ruderman N.B.
        Am. J. Physiol. 1994; 267: E95-E101
        • Reeves P.G.
        • Nielsen F.H.
        • Fahey Jr., G.C.
        J. Nutr. 1993; 123: 1939-1951
        • Ikemoto S.
        • Takahashi M.
        • Tsunoda N.
        • Maruyama K.
        • Itakura H.
        • Ezaki O.
        Metabolism. 1996; 45: 1539-1546
        • Mondon C.E.
        • Dolkas C.B.
        • Oyama J.
        Am. J. Physiol. 1981; 240: E482-E488
        • Cohen B.
        • Barkan D.
        • Levy Y.
        • Goldberg I.
        • Fridman E.
        • Kopolovic J.
        • Rubinstein M.
        J. Biol. Chem. 2001; 276: 7697-7700
        • Laborda J.
        Nucleic Acids Res. 1991; 19: 3998
        • Kushiro M.
        • Masaoka T.
        • Hageshita S.
        • Takahashi Y.
        • Ide T.
        • Sugano M.
        J. Nutr. Biochem. 2002; 13: 289-295
        • Kubota N.
        • Terauchi Y.
        • Miki H.
        • Tamemoto H.
        • Yamauchi T.
        • Komeda K.
        • Satoh S.
        • Nakano R.
        • Ishii C.
        • Sugiyama T.
        • Eto K.
        • Tsubamoto Y.
        • Okuno A.
        • Murakami K.
        • Sekihara H.
        • Hasegawa G.
        • Naito M.
        • Toyoshima Y.
        • Tanaka S.
        • Shiota K.
        • Kitamura T.
        • Fujita T.
        • Ezaki O.
        • Aizawa S.
        • Kadowaki T.
        Mol. Cell. 1999; 4: 597-609
        • Kliewer S.A.
        • Sundseth S.S.
        • Jones S.A.
        • Brown P.J.
        • Wisely G.B.
        • Koble C.S.
        • Devchand P.
        • Wahli W.
        • Willson T.M.
        • Lenhard J.M.
        • Lehmann J.M.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323
        • Memon R.A.
        • Tecott L.H.
        • Nonogaki K.
        • Beigneux A.
        • Moser A.H.
        • Grunfeld C.
        • Feingold K.R.
        Endocrinology. 2000; 141: 4021-4031
        • Motojima K.
        • Passilly P.
        • Peters J.M.
        • Gonzalez F.J.
        • Latruffe N.
        J. Biol. Chem. 1998; 273: 16710-16714
        • Tontonoz P.
        • Hu E.
        • Spiegelman B.M.
        Cell. 1994; 79: 1147-1156
        • Murakami K.
        • Tobe K.
        • Ide T.
        • Mochizuki T.
        • Ohashi M.
        • Akanuma Y.
        • Yazaki Y.
        • Kadowaki T.
        Diabetes. 1998; 47: 1841-1847
        • Rocchi S.
        • Picard F.
        • Vamecq J.
        • Gelman L.
        • Potier N.
        • Zeyer D.
        • Dubuquoy L.
        • Bac P.
        • Champy M.F.
        • Plunket K.D.
        • Leesnitzer L.M.
        • Blanchard S.G.
        • Desreumaux P.
        • Moras D.
        • Renaud J.P.
        • Auwerx J.
        Mol. Cell. 2001; 8: 737-747
        • Mukherjee R.
        • Hoener P.A.
        • Jow L.
        • Bilakovics J.
        • Klausing K.
        • Mais D.E.
        • Faulkner A.
        • Croston G.E.
        • Paterniti Jr., J.R.
        Mol. Endocrinol. 2000; 14: 1425-1433
        • Yamauchi T.
        • Waki H.
        • Kamon J.
        • Murakami K.
        • Motojima K.
        • Komeda K.
        • Miki H.
        • Kubota N.
        • Terauchi Y.
        • Tsuchida A.
        • Tsuboyama-Kasaoka N.
        • Yamauchi N.
        • Ide T.
        • Hori W.
        • Kato S.
        • Fukayama M.
        • Akanuma Y.
        • Ezaki O.
        • Itai A.
        • Nagai R.
        • Kimura S.
        • Tobe K.
        • Kagechika H.
        • Shudo K.
        • Kadowaki T.
        J. Clin. Investig. 2001; 108: 1001-1013
        • Lee H.J.
        • Choi S.S.
        • Park M.K.
        • An Y.J.
        • Seo S.Y.
        • Kim M.C.
        • Hong S.H.
        • Hwang T.H.
        • Kang D.Y.
        • Garber A.J.
        • Kim D.K.
        Biochem. Biophys. Res. Commun. 2002; 296: 293-299
        • Chakrabarti R.
        • Vikramadithyan R.K.
        • Misra P.
        • Hiriyan J.
        • Raichur S.
        • Damarla R.K.
        • Gershome C.
        • Suresh J.
        • Rajagopalan R.
        Br. J. Pharmacol. 2003; 140: 527-537
        • Wang Y.X.
        • Lee C.H.
        • Tiep S.
        • Yu R.T.
        • Ham J.
        • Kang H.
        • Evans R.M.
        Cell. 2003; 113: 159-170
        • Dressel U.
        • Allen T.L.
        • Pippal J.B.
        • Rohde P.R.
        • Lau P.
        • Muscat G.E.
        Mol. Endocrinol. 2003; 17: 2477-2493
        • Pereira M.A.
        • Jacobs Jr., D.R.
        • Pins J.J.
        • Raatz S.K.
        • Gross M.D.
        • Slavin J.L.
        • Seaquist E.R.
        Am. J. Clin. Nutr. 2002; 75: 848-855
        • Singh V.
        • Kaul S.
        • Chander R.
        • Kapoor N.K.
        Pharmacol. Res. 1990; 22: 37-44
        • Milligan S.
        • Kalita J.
        • Pocock V.
        • Heyerick A.
        • De Cooman L.
        • Rong H.
        • De Keukeleire D.
        Reproduction. 2002; 123: 235-242
        • Gerhauser C.
        • Alt A.
        • Heiss E.
        • Gamal-Eldeen A.
        • Klimo K.
        • Knauft J.
        • Neumann I.
        • Scherf H.R.
        • Frank N.
        • Bartsch H.
        • Becker H.
        Mol. Cancer Ther. 2002; 1: 959-969
        • Yasukawa K.
        • Takeuchi M.
        • Takido M.
        Oncology. 1995; 52: 156-158