help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1270
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/4/1640    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guo, Q.
Right arrow Articles by Wright, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guo, Q.
Right arrow Articles by Wright, S. D.
Endocrinology Vol. 145, No. 4 1640-1648
Copyright © 2004 by The Endocrine Society

A Novel Peroxisome Proliferator-Activated Receptor {alpha}/{gamma} Dual Agonist Demonstrates Favorable Effects on Lipid Homeostasis

Qiu Guo, Soumya P. Sahoo, Pei-Ran Wang, Denise P. Milot, Marc C. Ippolito, Margaret S. Wu, Joanne Baffic, Chhabi Biswas, Melba Hernandez, My-Hanh Lam, Neelam Sharma, Wei Han, Linda J. Kelly, Karen L. MacNaul, Gaochao Zhou, Ranjit Desai, James V. Heck, Thomas W. Doebber, Joel P. Berger, David E. Moller, Carl P. Sparrow, Yu-sheng Chao and Samuel D. Wright

Departments of Atherosclerosis and Endocrinology (Q.G., P.-R.W., D.P.M., M.C.I., J.B., M.H., M.-H.L., C.P.S., Y.-S.C., S.D.W.), Metabolic Disorders (M.W., C.B., N.S., L.J.K., K.L.M., G.Z., T.W.D., J.P.B., D.E.M.), and Medicinal Chemistry (S.P.S., W.H., R.D., J.V.H.), Merck Research Laboratories, Rahway, New Jersey 07065-0900

Address all correspondence and requests for reprints to: Qiu Guo, R80W250, P.O. Box 2000, Merck & Co. Inc., Rahway, New Jersey 07065. E-mail: qiu_guo{at}merck.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Patients with type 2 diabetes mellitus exhibit hyperglycemia and dyslipidemia as well as a markedly increased incidence of atherosclerotic cardiovascular disease. Here we report the characterization of a novel arylthiazolidinedione capable of lowering both glucose and lipid levels in animal models. This compound, designated TZD18, is a potent agonist with dual human peroxisome proliferator-activated receptor (PPAR)-{alpha}/{gamma} activities. In keeping with its PPAR{gamma} activity, TZD18 caused complete normalization of the elevated glucose in db/db mice and Zucker diabetic fatty rats. TZD18 lowered both cholesterol and triglycerides in hamsters and dogs. TZD18 inhibited cholesterol biosynthesis at steps before mevalonate and reduced hepatic levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Moreover, TZD18 significantly suppressed gene expression of fatty acid synthesis and induced expression of genes for fatty acid degradation and triglyceride clearance. Studies on 17 additional PPAR{alpha} or PPAR{alpha}/{gamma} agonists showed that lipid lowering in hamsters correlated with the magnitude of hepatic gene expression changes. Importantly, the presence of PPAR{gamma} agonism did not affect the relationship between hepatic gene expression and lipid lowering. Taken together, these data suggest that PPAR{alpha}/{gamma} agonists, such as TZD18, affect lipid homeostasis, leading to an antiatherogenic plasma lipid profile. Agents with these properties may provide favorable means for treatment of type 2 diabetes and dyslipidemia and the prevention of atherosclerotic cardiovascular disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES MELLITUS is a chronic disease characterized by glucose intolerance, hyperinsulinemia, and dyslipidemia (1). Blood glucose levels and insulin sensitivity can be improved by treating patients with currently marketed thiazolidinediones (TZDs) like rosiglitazone and pioglitazone (2). These agents act by binding and activating the transcription factor known as peroxisome proliferator-activated receptor (PPAR){gamma} (3, 4, 5). Whereas rosiglitazone and pioglitazone can markedly reduce insulin resistance (6, 7), they have modest effects on plasma lipids in type 2 diabetic patients (8, 9, 10). On the other hand, increased triglycerides and decreased high-density lipoprotein (HDL) cholesterol levels, the most common lipid abnormalities in patients with insulin resistance, can be effectively altered by treatment with fibrates such as fenofibrate, gemfibrozil, and bezafibrate (11, 12, 13). The fibrates act by binding and activating PPAR{alpha} (14). Both PPAR{gamma} agonists such as rosiglitazone and pioglitazone and PPAR{alpha} agonists such as fenofibrate have actions with distinct benefits for type 2 diabetes.

PPARs consist of three nuclear receptor isoforms, PPAR{alpha}, PPAR{gamma}, and PPAR{delta}, encoded by separate genes (for a recent review, see Ref. 15 and references therein). PPARs are ligand-dependent transcription factors that regulate expression of target genes related to lipid and glucose metabolism. Each PPAR forms a heterodimer with a retinoid X receptor that binds to specific peroxisome proliferator response elements. With the binding of a PPAR agonist and a subsequent conformational change in the receptor, coactivators are recruited to the PPAR/retinoid X receptor complex resulting in an augmentation in the initiation of gene transcription. The PPAR{alpha} receptor is expressed primarily in the liver and to a lesser degree in kidney, skeletal muscle, and cardiac muscle. It plays a critical role in the regulation of the cellular uptake, activation, and ß-oxidation of fatty acids (15, 16, 17). In contrast, the PPAR{gamma} receptor is predominantly expressed in adipose tissue in which it mediates transcriptional activation of genes involved in the regulation of lipid uptake and lipogenesis (15, 16). The physiologic role of the PPAR{delta} receptor is less well established. It has recently been reported that GW501516, a PPAR{delta} agonist, can induce a substantial increase in HDL-cholesterol levels as well as a reduction in triglyceride levels in obese Rhesus monkeys, but definitive evidence supporting a primary role for the human PPAR{delta} receptor as a key modulator of lipid metabolism has yet to be presented (18).

The overwhelming source of morbidity and mortality in type 2 diabetes is cardiovascular disease (19). There is thus an urgent medical need for the development of antidiabetic drugs that treat not only hyperglycemia but also dyslipidemia. The combined actions of PPAR{alpha} and PPAR{gamma} agonists appear ideally suited to this goal. Indeed, a PPAR{alpha}/{gamma} dual agonist, a TZD derivative called MK-767 (formerly KRP-297), has been shown to have in vivo efficacy in ameliorating insulin resistance in ob/ob and db/db mice and lowering plasma lipids in obese Zucker fatty rats (20, 21, 22, 23). Additional work has identified two structurally distinct, non-TZD agonists for PPAR{gamma} and PPAR{alpha} (24, 25). Both these compounds can improve hyperglycemia and hypertriglyceridemia in hyperglycemic db/db mice or Zucker diabetic fatty rats. In the present study, we characterized in detail the in vitro PPAR receptor-binding and cell-based transactivation properties of an additional TZD analog, compound TZD18 (compound 12 in Ref. 26) and characterized its ability to lower both lipids and glucose in three different animal models. In particular, our studies examined the lipid-lowering mechanisms of this compound and sought to determine whether its PPAR{gamma} activity affects lipid lowering mediated through its activation of PPAR{alpha}. Our results indicate that TZD18 has powerful antihyperlipidemic effects that are mediated by PPAR{alpha} regulated genes that are known to control hepatic lipid metabolism and that these PPAR{alpha} agonist activities are not compromised by the compound’s ability to also activate PPAR{gamma}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The TZDs, rosiglitazone [([±]-5-(4-[2-(methyl-2-pyridinylamino)ethoxy]phenyl)methyl]-2,4-thiazolidinedione] (4), and TZD18 [5-(3-[3-(4-phenoxy-2-propylphenoxy)propoxy]phenyl)-2,4-thiazolidinedione] (26); the non-TZDs, nTZD3 [3-(4-[3-phenyl-7-propyl-benzisoxazole-6-yl]oxy)butyloxyphenylacetic acid] (27) and nTZD4 [3-chloro-4-(3-[3-trifluoromethyl-7-propyl-benzisoxazole-6-yl]oxypropyl)thiophenylacetic acid] (27); fenofibrate (Sigma, St. Louis, MO); and simvastatin were used in this study. Additional 16 PPAR{alpha} or PPAR{alpha}/{gamma} agonists belonging to TZD or non-TZD were also used in this study. Cell culture reagents were obtained from Invitrogen (Carlsbad, CA).

Preparation of recombinant PPAR and binding assay
Human PPAR{alpha}, PPAR{gamma}, and PPAR{delta} receptors were expressed as recombinant glutathione-S-transferase (GST)-fusion proteins in Escherichia coli as previously described (28). The purified GST-hPPAR receptors were used in scintillation proximity assay (SPA)-based receptor-binding assays. Briefly, a human GST-PPAR receptor was combined in SPA buffer [10 mM Tris (pH 7.2), 1 mM EDTA, 10% glycerol, 10 mM sodium molybdate, 1 mM dithiothreitol, and 2 µg/ml benzamidine], 0.1% nonfat dry milk, 8.3 µg/ml anti-GST antibodies (Amersham Biosciences, Piscataway, NJ), and a radioligand in a total volume of 74 µl. For PPAR{alpha} and PPAR{gamma}, 5 nM of [3H2]nTZD3 (specific activity of 34.3 Ci/mmol) was added; for PPAR{delta}, 2.5 nM of [3H2]nTZD4 (specific activity of 13.4 Ci/mmol) was used. Yttrium silicate protein A-coated SPA beads (Amersham Biosciences) suspended in SPA buffer were added to a final concentration of 1.25 mg/ml. The assay plates in the presence of varying concentrations of TZD18 (in dimethyl sulfoxide) were incubated with shaking at 15 C for approximately 16 h. The plates were then counted in a TopCount scintillation counter (Packard Bioscience, Meriden, CT) to determine the displacement of the radioligand from the receptor by the compound. Nonspecific binding was determined by using a 100-fold excess of the respective unlabeled ligand. The results are expressed as IC50 calculated by a four-parameter logistic equation.

Cell culture and transactivation assay
COS-1 cells were cultured, and transactivation assays were carried out as previously described (27). Cells were cotransfected with pcDNA3-PPAR/GAL4 expression vector, pUAS(5X)-tk-luc reporter vector, and pCMV-lacZ as an internal control for transactivation efficiency using Lipofectamine (Invitrogen). Varying concentrations of TZD18 were incubated with the transfected cells at 37 C for 48 h. Cell lysates were then produced with reporter lysis buffer (Promega, Milwaukee, WI), and luciferase activity in cell extracts was determined by using luciferase assay buffer (Promega) in an ML3000 luminometer (Dynatech Laboratories, Chantilly, VA).

Animals
All animal study protocols were approved by the Merck Institutional Animal Care and Use Committee (Rahway, NJ) and were performed in AAALAC accredited facilities. Male db/db and nondiabetic db/+ (lean) mice (12–13 wk of age, Jackson Laboratories, Bar Harbor, ME) were housed (seven per box) and provided with ad libitum access to milled rodent chow (Purina 5008) and water. Mice were dosed daily by gavage with vehicle (0.5% methyl cellulose), TZD18 (3 mg/kg·d) or rosiglitazone (10 mg/kg·d) for 11 d. Blood was collected from the tail at d -4 and d 0 before treatment and 24 h after 4, 7, and 11 d treatment. Plasma levels of triglycerides were measured with an assay kit (Roche Diagnostics, Indianapolis, IN). Plasma glucose levels were determined with a glucose assay kit (Sigma).

Male beagle dogs weighing between 12 and 18 kg (Marshall Farm, North Rose, NY) were housed individually, fed a cholesterol-free chow diet ad libitum, and had free access to water. Before the start of experiments, the dogs were bled weekly from the jugular vein, and their serum cholesterol levels were determined. During the experiment, the dogs were treated by gavage with compounds suspended in 0.5% methyl cellulose for 2 wk. Blood samples were taken at the indicated days 24 h after the dosing periods. Serum cholesterol levels were determined enzymatically according to manufacturer’s instructions (WAKO Diagnostics, Richmond, VA).

Male golden Syrian hamsters weighing 100–120 g (Charles River Laboratory, Wilmington, MA) were housed (five per box), fed a commercial rodent diet, and given free access to water. Hamsters were gavaged with compounds in 0.5% methyl cellulose for the indicated days. On the last day, animals were euthanized with CO2, and serum samples were taken by heart puncture for lipid profile analysis. Liver samples were removed and immediately frozen in liquid nitrogen and stored at -70 C. Total plasma cholesterol and triglyceride levels were determined enzymatically as described above.

Serum lipoproteins were separated by a fast protein liquid chromatography (FPLC) system using a Superose 6 HR column (Amersham Pharmacia Biotech, Piscataway, NJ). The column was equilibrated with PBS (pH 7.4) plus 1 mM EDTA and was run at a flow rate of 0.2 ml/min. The serum samples were pooled (three animals per pool), filtered with 0.65 µm microcentrifuge filters, and then 200 µl samples were loaded onto the column. Fractions (0.27 ml) were collected and analyzed for total cholesterol concentrations as described above.

Inhibition of in vivo cholesterol synthesis
Inhibition of cholesterol synthesis in hamsters was determined by orally dosing the animals with the indicated compounds and measuring the incorporation of 14C-acetate or 14C-mevalonate into hepatic cholesterol (29). Hamsters were maintained under reverse lighting conditions and orally dosed daily with the compounds for 9 d. On the 10th day, each hamster was intraperitoneally injected with 12 µCi of 14C-acetate or 14C-mevalonate (Sigma). The animals were euthanized 1 h after the injection. One gram of the liver samples was sliced, hydrolyzed with 5 ml alcoholic KOH, and then incubated for 3 h at 70 C. The liver cholesterol was extracted using 5 ml water and 10 ml petroleum ether. Six milliliters of petroleum ether were evaporated under nitrogen and dissolved with 5 ml of the BioSafe-II scintillation cocktail. The incorporation of 14C into the cholesterol was determined by liquid scintillation counting.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity assay
Approximately 1 g of the frozen liver from vehicle and compound-treated hamsters was used in preparation of microsomes at 4 C as a source of HMG-CoA reductase (30, 31). HMG-CoA reductase activity was determined as previously described (30, 31). The protein concentrations were measured by the method of Lowry et al. (32).

Real-time quantitative PCR analysis
TaqMan PCR analysis was used to determine the relative mRNA expression levels of acetyl CoA carboxylase (ACC), acyl CoA oxidase (ACO), apolipoprotein C-III (apo C-III), and lipoprotein lipase (LPL) as previously described (33). Oligonucleotide primers and TaqMan probes were designed using Primer Expression 1.0 (Applied Biosystems, Foster City, CA) for ACC (accession no. AF356089), apo C-III (AF356088), ACO (AF356085), and LPL (AF356087) (see Table 1Go). Total RNA was isolated from livers (five animals per group) using Trizol (Invitrogen) according to the manufacturer’s instructions. The RNA was then treated with RQ1 RNase-free DNase (Promega) at 37 C for 60 min, followed by ethanol precipitation, to eliminate the contaminating genomic DNA in the RNA samples. RT-PCR were carried out at 25 C for 10 min, 48 C for 30 min, and 95 C for 5 min in a 96-well plate using TaqMan reverse transcription reagents (Applied Biosystems). The TaqMan assay was performed in 25 µl containing 25 ng cDNA, 300 nM primers, and 200 nM FAM-labeled TaqMan probes for the target genes, 80 nM of both the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers and VIC-labeled TaqMan probe, and the TaqMan universal PCR master mix. Experiments were performed in duplicate. Relative quantitation of mRNA expression levels (compound-treated animals/vehicle ratio) was calculated by comparing the target gene/GAPDH of the treated animals to those of the vehicles.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Hamster primers and probe sets used for TaqMan real-time PCR

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TZD18 is a novel dual PPAR{alpha}/{gamma} agonist
Using a combination of directed chemical synthesis and molecular modeling, we synthesized a series of 5-aryl thiazolidine-2,4-diones compounds for identification of novel dual ligands for human PPAR{alpha} and human PPAR{gamma} receptors with minimal human PPAR{delta} receptor-binding activity (26). A radioligand-binding assay was used for screening novel PPAR{alpha}, PPAR{gamma}, or dual PPAR{alpha}/{gamma} ligands (28). In this ligand-displacement assay, the ability of test compounds to displace binding of a potent PPAR{gamma}/{alpha} ligand, [3H2]-TZD3, from recombinant human PPAR{gamma} was measured (28). A similar protocol was applied to evaluate binding of test compounds to human PPAR{alpha} once again using [3H2]nTZD3. Ligand displacement identified TZD18 as a potent human PPAR{alpha} ligand with a IC50 of 28 nM, as compared with an IC50 of 35 µM for the human PPAR{alpha} agonist fenofibrate (34). TZD18 also exhibited 10-fold higher potency for human PPAR{gamma} than the antidiabetic PPAR{gamma} agonist rosiglitazone (IC50 = 57 nM vs. 568 nM, respectively; see Table 2Go) (4, 5). In contrast, TZD18 had essentially no binding activity to human PPAR{delta} (IC50 > 10 µM). Thus, TZD18 is a newly discovered, human PPAR{alpha}/{gamma} dual ligand (see Fig. 1Go).


View this table:
[in this window]
[in a new window]
  		TABLE 2. In vitro PPAR binding and transactivation activities of TZD18, rosiglitazone, and fenofibric acid

 


View larger version (9K):
[in this window]
[in a new window]
  		FIG. 1. Structure of the dual PPAR{alpha}/{gamma} agonist TZD18.

 
To examine whether TZD18 can act as an agonist of both PPAR{alpha} and PPAR{gamma} receptors, we used cell-based transactivation assays to test its ability to activate PPAR{alpha}/GAL4 and PPAR{gamma}/GAL4 transcriptional regulation of the pUAS(5X)-tk-luciferase (27). Series of dilutions of TZD18 was incubated with transiently transfected COS-1 cells at 37 C for 48 h and luciferase activity in cell extracts determined. TZD18 activated human PPAR{alpha} and PPAR{gamma} in dose-dependent manners with EC50 of 45 and 20 nM, respectively (Table 2Go). TZD18 also showed comparable activities on hamster, mouse, and dog PPAR{gamma} (EC50 = 33, 77, and 50 nM, respectively). The agonist activity of the compound on the dog PPAR{alpha} receptor was similar to that on the human PPAR{alpha} receptor with an EC50 value of 34 nM, whereas its hamster PPAR{alpha} receptor activity was approximately 6-fold lower than that on human. Interestingly, TZD18 has no significant activity on the mouse PPAR{alpha} receptor. Thus, the TZD18 ligand can potently activate both human PPAR{alpha} and PPAR{gamma} receptors.

Effects of TZD18 on elevated glucose and lipid levels in obese rodent models
PPAR{gamma} agonists, including rosiglitazone, exhibit substantial insulin sensitizing effects in the db/db mouse, an obese animal model of type 2 diabetes (4, 5). Given that TZD18 is a PPAR{gamma} agonist and has excellent pharmacokinetic properties (26), we surmised that this compound should be efficacious in db/db mice. To directly test this hypothesis, we examined the in vivo activity of TZD18 by treating db/db mice with this compound at 3 mg/kg·d or rosiglitazone at 10 mg/kg·d for 11 d. In this experiment, nondiabetic db/+ (lean) mice served as a control. Over the course of 11 d, marked hyperglycemia in db/db mice was gradually reduced by daily treatment with TZD18 (Fig. 2AGo). The extent of correction of hyperglycemia in db/db mice relative to vehicle control was 91% after 11 d. At this point, the levels of plasma glucose were close to those of the normal db/+ lean mice. By comparison, rosiglitazone at 10 mg/kg·d resulted in a 69% correction of hyperglycemia. TZD18, therefore, is more potent than rosiglitazone for normalizing hyperglycemia in this model.



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 2. Effects of TZD18 and rosiglitazone on hyperglycemia (A) and hypertriglyceridemia (B) in db/db mice. db/db mice (n = 7 mice per group) were orally dosed with vehicle (0.5% methyl cellulose, open diamonds), TZD18 (3 mg/kg·d, open circles), or rosiglitazone (10 mg/kg·d, solid squares) for 11 d. Solid triangles correspond to nondiabetic db/+ mice control. Plasma glucose and triglyceride levels were measured 24 h after the dosing as described in Materials and Methods.

 
We also investigated the effects of TZD18 on plasma triglyceride levels in the db/db mouse because this animal exhibits hypertriglyceridemia. Plasma triglyceride levels in db/db mice are also normalized with TZD18 treatment at both oral doses of 3 and 10 mg/kg·d, with 64 and 79% reductions in plasma triglyceride levels, respectively (Fig. 2BGo; data not shown). In the corresponding study, rosiglitazone at 10 mg/kg·d reduced plasma triglyceride levels by 69% (Fig. 2BGo). Our results clearly indicate that daily administration of TZD18 to diabetic db/db mice resulted in nearly complete correction of elevated plasma levels of both glucose and triglycerides.

Effects of TZD18 on plasma cholesterol and triglyceride levels in dogs
The dog exhibits strong cholesterol lowering in response to statins and was used as the principal preclinical species during the development of lovastatin and simvastatin. The dog was therefore chosen as a key species for testing the lipid lowering efficacy of TZD18. Oral administration of TZD18 to dogs led to a large reduction in serum cholesterol. The average serum cholesterol decreased 10, 24, and 34% in dogs after 14 d at 1, 3, and 5 mg/kg·d TZD18, respectively, compared with a 16% decrease by simvastatin at 4 mg/kg·d (Fig. 3Go). For comparison, fenofibrate at 100 mg/kg·d was found to have similar cholesterol-lowering effects as TZD18 at 3 mg/kg·d (data not shown). To address whether TZD18 and simvastatin can exert an additive effect in lowering cholesterol, dogs were treated with a combination of TZD18 at 3 mg/kg·d and simvastatin at 4 mg/kg·d. A 35% reduction in serum cholesterol levels was observed. Taken together, our results indicate that TZD18 potently lowers serum cholesterol in dogs and can be used in combination with simvastatin to efficaciously treat dyslipidemia.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 3. Time course of serum cholesterol lowering by TZD18 and fenofibrate in dogs. Dogs (n = 5 per group) were treated by gavage with vehicle (open diamonds), TZD18 at 1 mg/kg·d (open triangles), 3 mg/kg·d (solid triangles), 5 mg/kg·d (open circles), simvastatin at 4 mg/kg·d (solid squares), and combination of TZD18 at 3 mg/kg·d and simvastatin at 4 mg/kg·d (solid circles) for 14 d. Blood samples were taken at the indicated days 24 h after administration of a dose, and serum cholesterol levels were determined. Data are expressed as mean ± SE for five animals in relative units taking the average values from respective group determined 2 wk before the treatment as 100%. Control values for the different groups ranged from 150 ± 29 to 163 ± 12 mg dl-1 for cholesterol levels.

 
Effects of TZD18 on serum lipid levels in hamsters
To further characterize the lipid effects of TZD18, we turned to the hamster. Oral administration of TZD to chow-fed hamsters for 9 d led to dose-dependent decreases in serum triglycerides (Fig. 4BGo). At 10 mg/kg·d, the serum triglyceride concentrations in the treated groups were 27 and 7% lower for TZD18 and fenofibrate, respectively, than those in the control group. Whereas the 100 mg/kg·d dose of fenofibrate brought triglyceride concentrations down by 31%, 3 mg/kg·d of treatment with TZD18 reduced triglycerides by 24%. TZD18 thus showed approximately 3- to 10-fold greater dose potency than fenofibrate, a PPAR{alpha} agonist devoid of PPAR{gamma} activity. Treatment with TZD18 also caused dose-dependent cholesterol lowering, with a maximal cholesterol lowering of 60% achieved at 30 mg/kg·d (Fig. 4AGo). In contrast, even at a dose as high as 300 mg/kg·d, fenofibrate lowered cholesterol by only 40% (Fig. 4AGo). Therefore, TZD18 shows both superior dose potency and greater maximal effects on cholesterol than fenofibrate. It is possible that the PPAR{gamma} activity of TZD18 contributes to this potency because treatment of hamsters with rosiglitazone at 10 mg/kg·d decreased cholesterol approximately 15% (data not shown). However, the contribution of PPAR{gamma} activity is likely to be low.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 4. Dose-dependent lowering of serum lipids in TZD18 or fenofibrate-treated hamsters fed with a normal chow diet. Hamsters were treated with TZD18 (open circles) or fenofibrate (open squares) at the indicated doses for 9 d. Serum cholesterol (A) and triglyceride (B) levels were then determined at the 10th day 24 h after dosing. Data are expressed as mean ± SE for 10 animals in relative units taking the vehicle controls as 100%. Control values for the different experiments ranged from 94 ± 2 to 133 ± 5 mg dl-1 for cholesterol and 229 ± 9 to 268 ± 29 mg dl-1 for triglycerides. C, Distribution of cholesterol from serum of vehicle controls and TZD18-treated hamsters. Animals were treated with TZD18 at 10 mg/kg·d for 9 d. Serum lipoprotein cholesterol distributions of vehicle (solid diamonds) and TZD18-treated hamsters (open circles) were analyzed by FPLC. The eluted fractions (0.27 ml/fraction) were assessed for cholesterol concentrations. Data are expressed as mean ± SE of three groups of serum (three animals per group).

 
To further analyze the distribution of serum lipoproteins from TZD18-treated hamsters (10 mg/kg·d), aliquots from pooled sera were size-fractionated by FPLC. The cholesterol profiles showed that treatment with TZD18 caused a decrease in HDL (44%), low-density lipoprotein (39%), and very low-density lipoprotein (22%) levels (Fig. 4CGo). To determine whether the reduction in serum HDL-cholesterol in response to TZD18 is correlated with the regulation of apolipoprotein A-I (apo A-I) expression in the hamster liver (35, 36), we evaluated apo A-I gene expression in these animals. With the TZD18 treatment, hamsters showed a 61% reduction in hepatic apo A-I mRNA levels, suggesting that TZD18 can lower serum HDL-cholesterol by down-regulating hamster liver apo A-I expression. Interestingly, a comparable extent of the apo A-I transcription reduction after fenofibrate treatment was reported in mice (35), whereas an opposite effect of fenofibrate is observed in transcription of human apo A-I (35).

Effects of TZD18 on hepatic cholesterol biosynthesis
To understand the molecular basis for TZD18-mediated lipid lowering in vivo, we characterized the effects of TZD18 on hepatic cholesterol biosynthesis in the hamster by measuring the in vivo rates of incorporation of 14C-acetate or 14C-mevalonate into hepatic cholesterol (see Materials and Methods). As shown in Fig. 5AGo, the incorporation of 14C-acetate into hepatic cholesterol was decreased significantly, reaching a 53% reduction at 10 mg/kg·d of TZD18. In comparison, simvastatin at 8 mg/kg·d and fenofibrate at 150 mg/kg·d caused reductions of 53 and 75%, respectively. In contrast, hepatic cholesterol synthesis from 14C-mevalonate was not appreciably inhibited by any of the treatment regimens (Fig. 5BGo). These results suggest that TZD18 acts on the cholesterol biosynthetic pathway at the level of the enzymatic conversions between acetyl-CoA and mevalonate, as do fenofibrate and simvastatin. This mechanism of action is also shared by fenofibrate, which is an agonist only for the PPAR{alpha} receptor, so that the PPAR{alpha} activity likely serves a crucial role in inhibiting cholesterol biosynthesis.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 5. TZD18 inhibits hepatic cholesterol synthesis in hamsters. Hamsters were maintained under a reverse lighting room and dosed with the indicated compounds for 9 d. Hepatic cholesterol synthesis was measured by ip injecting with 12.5 µCi of 14C-acetate (A) or 14C-mevalonate (B) into the hamsters and after the incorporation of 14C in the cholesterol 1 h after the injection as described in Materials and Methods. *, P < 0.05.

 
Suppression of HMG-CoA reductase activities in hamsters by TZD18
We have previously shown that fenofibrate blocks cholesterol biosynthesis by lowering the activity of HMG-CoA reductase (33), a key enzyme in the cholesterol biosynthetic pathway (reviewed in Ref. 37). We hypothesized that TZD18 altered cholesterol biosynthesis in a similar manner. To test this, we characterized the effects of TZD18 on HMG-CoA reductase activity in hamster liver microsomes purified from vehicle controls and TZD18-treated animals. In the controls, hepatic microsome HMG-CoA reductase activity was 32 ± 6 pmol 14C-mevalonate synthesized per milligram protein per minute. TZD18 and fenofibrate decreased the reductase activity by 22 and 20%, respectively, relative to the control group (Fig. 6Go). Thus, TZD18, like fenofibrate, suppresses cholesterol synthesis by reducing hepatic HMG-CoA reductase activity.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6. Reduction of hepatic microsomal HMG-CoA reductase activities in TZD18 or fenofibrate-treated hamsters. Hamsters were treated with vehicle (open diamonds), TZD18 at 30 mg/kg·d (open circles) or fenofibrate at 100 mg/kg·d (solid squares) for 7 d under a normal chow diet. At the eighth day, the hamsters were euthanized, and the livers were quickly frozen. The hepatic HMG CoA reductase activities were measured in triplicate from microsomes isolated from the livers as described in Materials and Methods.

 
Effects of TZD18 on the expression of genes involved in triglyceride metabolism
To further define the mechanisms by which TZD18 lowers triglyceride levels, we investigated the effects of TZD18 on the expression of genes involved in triglyceride metabolism. We orally dosed hamsters for 9 d with compounds and then determined the relative hepatic expression levels of these genes using TaqMan real-time PCR analysis. Expression of ACC and apo C-III mRNA were reduced to 72 and 34%, respectively, of the control after TZD18 treatment (Fig. 7Go). In the same experiment, TZD18 caused 6.3- and 5.2-fold increases, respectively, in LPL and ACO mRNA levels. As we have previously shown (33), fenofibrate had similar effects on the expression of the four genes described above (Fig. 7Go). In contrast, rosiglitazone only weakly affected the expression of these genes (Fig. 7Go). These results reinforce the notion that the presence of PPAR{gamma} agonist activity in TZD18 has relatively little influence on hepatic lipid metabolism in nondiabetic animals.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 7. Effects of TZD18 and fenofibrate on mRNA expression of ACC, LPL, ACO, and apo C-III in hamster livers. Hamsters were gavaged with vehicle (open diamonds), TZD18 at 30 mg/kg·d (open circles), fenofibrate at 150 mg/kg·d (open squares), and rosiglitazone at 10 mg/kg·d (open triangles) for 9 d. At 10 d, the hamsters were euthanized, and the livers were immediately frozen. Total RNA was isolated from the livers. The relative mRNA expression levels of the genes were measured by TaqMan PCR analysis. Data are expressed as the fold change and 95% confidence interval [mean (lower limit, upper limit)] normalized to GAPDH mRNA expression, in which the value for vehicle was set at 1.00. The data were performed in duplicate from five animals. Statistically (via ANOVA method) significant difference between compound-treated and vehicles at P < 0.001 are indicated by two asterisks and P < 0.05 by one asterisk.

 
Relation of hepatic mRNA levels to serum triglyceride reduction in hamsters treated with PPAR agonists
To confirm the role of hepatic gene expression changes in mediating the lipid-lowering effects of PPAR{alpha} agonists, a variety of structurally diverse PPAR{alpha} agonists were administered to hamsters and changes in gene expression were plotted vs. extent of triglyceride lowering (Fig. 8Go, open triangles). We found a strong positive correlation between liver LPL mRNA levels and triglyceride-lowering (Pearson correlation coefficient = 0.76, P = 0.0002) and a weak negative correlation between liver apo C-III mRNA levels and triglyceride-lowering (Pearson correlation coefficient = 0.49, P = 0.04). Although there was no obvious correlation for ACC, in almost all cases, ACC mRNA levels were down-regulated by the compounds (data not shown). These results are consistent with the idea that changes in hepatic gene expression play a prime role in lipid lowering. To determine whether the presence of PPAR{gamma} agonism affects the relationship between hepatic gene expression and lipid lowering, parallel studies were performed with a variety of structurally diverse PPAR{alpha}/{gamma} dual agonists (Fig. 8Go, solid triangles). PPAR{gamma} activity has little consistent effect on the relationship of LPL or apo C-III mRNA levels to serum triglyceride decrease. This observation strongly suggests that the presence of PPAR{gamma} agonism neither enhances nor reduces the lipid lowering effect of PPAR{alpha} agonism.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 8. Relation of relative hepatic mRNA levels and percentage change of serum triglyceride levels in hamsters treated with either PPAR{alpha} agonists or dual PPAR{alpha}/{gamma} agonists. A, LPL. B, Apo C-III. Hamsters were treated with one of either PPAR{alpha} agonists (open triangles) or dual PPAR{alpha}/{gamma} agonists (solid triangles) for 9 d. Hamsters were euthanized 24 h after the last dosing. Serum triglyceride levels were determined. Relative hepatic mRNA levels of the genes were measured by TaqMan PCR analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Type 2 diabetes is a serious and growing health threat in the industrialized world. The medical management of diabetic patients often focuses on plasma glucose levels because it is believed that elevated glucose levels increase the risk for retinopathy, kidney disease, and peripheral neuropathy. Diabetic patients are also at high risk for atherosclerosis and myocardial infarction, and some of this risk can probably be attributed to diabetic dyslipidemia, which is characterized by high triglyceride and low HDL levels (for a recent review, see Ref. 19 and references therein). The PPAR{gamma} agonists rosiglitazone and pioglitazone are currently in clinical use for lowering glucose levels in diabetics. Rosiglitazone does not significantly improve lipid levels in diabetics (9), whereas pioglitazone, which has modest PPAR{alpha} activity, has been reported to lower triglyceride levels up to 16% and to raise HDL-cholesterol up to 20% (8).

In both diabetic and nondiabetic patients, hypertriglyceridemia is often treated with fibrates, which are PPAR{alpha} agonists. Fibrates can lower triglyceride levels 30–50%, and also increased HDL up to 20% but do not improve glucose levels. There might be major clinical utility, therefore, for novel therapeutic agents that can correct both hyperglycemia and diabetic dyslipidemia. The present work describes such an agent: TZD18, a PPAR{alpha}/{gamma} dual agonist.

We characterized TZD18 in multiple animal models. TZD18 displayed robust antidiabetic activity in db/db mice and Zucker diabetic fatty rats (data not shown) and was more potent than rosiglitazone in both species. PPAR{gamma} agonists lower triglyceride much more effectively in db/db mice than they do in type 2 diabetic humans. The reasons for this discrepancy are not fully known, although one clear difference between db/db mice and typical type 2 diabetic patients is that the former have no leptin signaling. The db/db mouse model is a useful one, but no single animal model accurately reflects all facets of human metabolic disease. The imperfections in each animal model led us to test our investigational compound in multiple models in multiple preclinical species.

The lipid-lowering properties of TZD18 were also characterized in two species, the hamster and the dog. A perfect preclinical model for studying lipid metabolism does not exist (for a review, see Ref. 38). The mouse would have been an inappropriate choice for studying TZD18 because this agent lacks mouse PPAR{alpha} agonist activity and because cholesterol metabolism in rodents is dramatically different from that in man. The dog is the only model species in which statins, fibrates, and bile acid-binding resins all have the same effects on cholesterol and triglyceride metabolism as are seen in humans (Chao, Y.-S., unpublished observations). When administered to dogs, TZD18 potently lowered plasma cholesterol levels, an effect that was additive with simvastatin. The significance of coadministration with simvastatin will be discussed below.

Hamsters were used to further characterize the lipid-lowering actions of TZD18 and correlate these effects with alterations in the expression of genes known to control hepatic lipid metabolism. TZD18 lowered both cholesterol and triglycerides in hamsters in a manner that was both more potent and more efficacious than fenofibrate. The cholesterol-lowering effects of these PPAR ligands may be attributable to the observed decrease in hepatic microsomal HMG-CoA reductase activity that resulted in a diminution in cholesterol biosynthesis. Our gene expression studies support the conclusion that the triglyceride-lowering effects of TZD18 are probably mediated by as many as three distinct PPAR{alpha}-regulated hepatic pathways. First, the compound may inhibit an initial step in fatty acid synthesis catalyzed by ACC by lowering expression of this enzyme. Second, the compound may enhance the peroxisomal ß-oxidation of fatty acids by increasing expression of ACO, the rate-limiting enzyme in this process, and thereby increase triglyceride degradation. Third, TZD18 may lower triglyceride levels by increasing expression of LPL while decreasing that of apo C-III. Interestingly, among the four genes described above, it was the induction of LPL mRNA expression by TZD18 that correlated best with its triglyceride-lowering activity. The promoter of the gene encoding LPL has been shown to possess a bona fide peroxisome proliferator response element (39), and it has previously been shown that LPL expression is up-regulated in liver by PPAR{alpha} agonists and in adipose tissue by PPAR{gamma} agonists (16, 40). Although the liver is not usually considered a major source of lipoprotein lipase, even modest expression of this protein might influence triglyceride-rich lipoprotein secretion by catalyzing the reuptake of nascent particles (41).

An important consideration for PPAR{alpha}/{gamma} dual agents is whether the presence of PPAR{gamma} activity will affect the ability of PPAR{alpha} to achieve the expected effects on lipids. By acting on adipose tissue, PPAR{gamma} might affect the disposition of fatty acids so as to alter hepatic function. Our studies used a spectrum of PPAR{alpha} and PPAR{alpha}/{gamma} compounds to demonstrate that lipid lowering correlates strongly with the extent of PPAR{alpha}-driven changes in hepatic gene expression. This result was not unexpected due to the high level of hepatic PPAR{alpha} expression and the known primary role played by the liver in the maintenance of lipid homeostasis. In addition, our results suggested that the correlation of plasma lipid changes with hepatic gene expression changes is not affected by the addition of PPAR{gamma} agonism. This result suggests that the lipid-lowering actions of TZD18 and other PPAR{alpha}/{gamma} dual agonists might be well predicted by their ability to drive hepatic gene expression via PPAR{alpha}.

PPAR{alpha}/{gamma} dual agonists have been previously described by other investigators (21, 23, 24, 25). These three groups showed that their compounds were antidiabetic, but few data were published on corresponding lipid effects. Sauerberg et al. (24) report only a single experiment showing their compound reduced triglyceride and total serum cholesterol levels in cholesterol-fed rats. Etgen et al. (25) showed only that their compound increased HDL-cholesterol levels in human apo A-I transgenic mice, a result that might be confounded by the fact that their compound also had significant PPAR{delta} activity. Neither group reported extensive data on the intrinsic PPAR activity of their compounds in species other than humans, which limits the ability to interpret the potency of their compounds in small animals. The present work expands on the existing literature by demonstrating that the potent PPAR{alpha}/{gamma} dual-agonist TZD18 has lipid-lowering effects in the hamster and dog and by characterizing the changes in expression of critical genes involved in hepatic lipid metabolism.

The cholesterol-lowering effect of TZD18 was additive with that of simvastatin in dogs. This observation is important because of the recent finding that simvastatin decreases the risk of major coronary events in diabetic patients (42). It is likely, therefore, that there will be an increase in the number of diabetic patients taking simvastatin. Although there is an unfavorable drug-drug pharmacokinetic interaction between statins and gemfibrozil, this problem will probably not extend to new potent PPAR agonists. A major mechanism for the interaction appears to be competitive inhibition of the glucuronidation of statins by the extremely high concentrations of gemfibrozil required for lipid lowering (43). Blockade of glucuronidation is less likely with potent drugs that will be dosed at lower levels and is made still more unlikely by the absence in TZDs such as TZD18 of a carboxyl moiety that could serve as a target for glucuronidation. Thus, the coadministration of novel PPAR{alpha}/{gamma} dual agonists with simvastatin has the potential to decrease multiple risk factors for morbidity and mortality in diabetic patients and improve the quality and length of their lives.


   Acknowledgments
 
We thank Beverly Shelton for help with some of the animal work; Donghui Zhang for statistical advice on the data analysis; and Peter Meinke, John Menke, and Diane Shevell for critical reading of the manuscript.


   Footnotes
 
Abbreviations: ACC, Acetyl-CoA carboxylase; ACO, acyl-CoA oxidase; apo A-I, apolipoprotein A-I; apo C-III, apolipoprotein C-III; FPLC, fast protein liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; HDL, high-density lipoprotein; HMG CoA reductase, 3-hydroxy-3-methylglutaryl coenzyme A reductase; LPL, lipoprotein lipase; PPAR, peroxisome proliferator-activated receptor; SPA, scintillation proximity assay; TZD, thiazolidinedione.

Received September 23, 2003.

Accepted for publication December 23, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Reaven GM 1988 Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595–1607[Abstract]
  2. Olefsky JM, Saltiel AR 2000 PPAR{gamma} and the treatment of insulin resistance. Trends Endocrinol Metab 11:362–368[CrossRef][Medline]
  3. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  4. Willson TM, Cobb JE, Cowan DJ, Wiethe RW, Correa ID, Prakash SR, Beck KD, Moore LB, Kliewer SA, Lehmann JM 1996 The structure-activity relationship between peroxisome proliferator-activated receptor {gamma} agonism and the antihyperglycemic activity of thiazolidinediones. J Med Chem 39:665–668[CrossRef][Medline]
  5. Berger J, Bailey P, Biswas C, Cullinan CA, Doebber TW, Hayes NS, Saperstein R, Smith RG, Leibowitz MD 1996 Thiazolidinediones produce a conformational change in peroxisomal proliferator-activated receptor-{gamma}: binding and activation correlate with antidiabetic actions in db/db mice. Endocrinology 137:4189–4195[Abstract]
  6. Aronoff S, Rosenblatt S, Braithwaite S, Egan JW, Mathisen AL, Schneider RL 2000 Pioglitazone hydrochloride monotherapy improves glycemic control in the treatment of patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. The Pioglitazone 001 Study Group. Diabetes Care 23:1605–1611[Abstract/Free Full Text]
  7. Saltiel AR, Olefsky JM 1996 Thiazolidinediones in the treatment of insulin resistance and type II diabetes. Diabetes 45:1661–1669[Abstract]
  8. Herz M, Johns D, Reviriego J, Grossman LD, Godin C, Duran S, Hawkins F, Lochnan H, Escobar-Jimenez F, Hardin PA, Konkoy CS, Tan MH 2003 A randomized, double-blind, placebo-controlled, clinical trial of the effects of pioglitazone on glycemic control and dyslipidemia in oral antihyperglycemic medication-naive patients with type 2 diabetes mellitus. Clin Ther 25:1074–1095[CrossRef][Medline]
  9. Khan MA, St Peter JV, Xue JL 2002 A prospective, randomized comparison of the metabolic effects of pioglitazone or rosiglitazone in patients with type 2 diabetes who were previously treated with troglitazone. Diabetes Care 25:708–711[Abstract/Free Full Text]
  10. Miyazaki Y, Glass L, Triplitt C, Matsuda M, Cusi K, Mahankali A, Mahankali S, Mandarino LJ, DeFronzo RA 2001 Effect of rosiglitazone on glucose and non-esterified fatty acid metabolism in type II diabetic patients. Diabetologia 44:2210–2219[CrossRef][Medline]
  11. 1998 Choice of lipid-lowering drugs. Med Lett Drugs Ther 40:117–122
  12. Watts GF, Dimmitt SB 1999 Fibrates, dyslipoproteinaemia and cardiovascular disease. Curr Opin Lipidol 10:561–574[CrossRef][Medline]
  13. Rubins HB, Robins SJ, Collins D, Nelson DB, Elam MB, Schaefer EJ, Faas FH, Anderson JW 2002 Diabetes, plasma insulin, and cardiovascular disease: subgroup analysis from the Department of Veterans Affairs high-density lipoprotein intervention trial (VA-HIT). Arch Intern Med 162:2597–2604[Abstract/Free Full Text]
  14. Issemann I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645–650[CrossRef][Medline]
  15. Berger J, Moller DE 2002 The mechanisms of action of PPARs. Annu Rev Med 53:409–435[CrossRef][Medline]
  16. Schoonjans K, Staels B, Auwerx J 1996 The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta 1302:93–109[Medline]
  17. Schoonjans K, Staels B, Auwerx J 1996 Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J Lipid Res 37:907–925[Abstract]
  18. Oliver Jr WR, Shenk JL, Snaith MR, Russell CS, Plunket KD, Bodkin NL, Lewis MC, Winegar DA, Sznaidman ML, Lambert MH, Xu HE, Sternbach DD, Kliewer SA, Hansen BC, Willson TM 2001 A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport. Proc Natl Acad Sci USA 98:5306–5311[Abstract/Free Full Text]
  19. Peter P, Nuttall SL, Kendall MJ 2003 Insulin resistance—the new goal! J Clin Pharm Ther 28:167–174[CrossRef][Medline]
  20. Calkin AC, Thomas MC, Cooper ME 2003 MK-767. Kyorin/Banyu/Merck. Curr Opin Investig Drugs 4:444–448[Medline]
  21. Murakami K, Tobe K, Ide T, Mochizuki T, Ohashi M, Akanuma Y, Yazaki Y, Kadowaki T 1998 A novel insulin sensitizer acts as a coligand for peroxisome proliferator-activated receptor-{alpha} (PPAR-{alpha}) and PPAR-{gamma}: effect of PPAR-{alpha} activation on abnormal lipid metabolism in liver of Zucker fatty rats. Diabetes 47:1841–1847[Abstract]
  22. Murakami K, Tsunoda M, Ide T, Ohashi M, Mochizuki T 1999 Amelioration by KRP-297, a new thiazolidinedione, of impaired glucose uptake in skeletal muscle from obese insulin-resistant animals. Metabolism 48:1450–1454[CrossRef][Medline]
  23. Nomura M, Kinoshita S, Satoh H, Maeda T, Murakami K, Tsunoda M, Miyachi H, Awano K 1999 (3-substituted benzyl)thiazolidine-2, 4-diones as structurally new antihyperglycemic agents. Bioorg Med Chem Lett 9:533–538[CrossRef][Medline]
  24. Sauerberg P, Pettersson I, Jeppesen L, Bury PS, Mogensen JP, Wassermann K, Brand CL, Sturis J, Woldike HF, Fleckner J, Andersen AS, Mortensen SB, Svensson LA, Rasmussen HB, Lehmann SV, Polivka Z, Sindelar K, Panajotova V, Ynddal L, Wulff EM 2002 Novel tricyclic-{alpha}-alkyloxyphenylpropionic acids: dual PPAR{alpha}/{gamma} agonists with hypolipidemic and antidiabetic activity. J Med Chem 45:789–804[CrossRef][Medline]
  25. Etgen GJ, Oldham BA, Johnson WT, Broderick CL, Montrose CR, Brozinick JT, Misener EA, Bean JS, Bensch WR, Brooks DA, Shuker AJ, Rito CJ, McCarthy JR, Ardecky RJ, Tyhonas JS, Dana SL, Bilakovics JM, Paterniti Jr JR, Ogilvie KM, Liu S, Kauffman RF 2002 A tailored therapy for the metabolic syndrome: the dual peroxisome proliferator-activated receptor-{alpha}/{gamma} agonist LY465608 ameliorates insulin resistance and diabetic hyperglycemia while improving cardiovascular risk factors in preclinical models. Diabetes 51:1083–1087[Abstract/Free Full Text]
  26. Desai R, Han W, Metzger EJ, Bergman JP, Gratale DF, MacNaul KL, Berger JP, Doebber TW, Leung K, Moller DE, Heck JV, Sahoo SP 2003 5-Aryl thiazolidine-2, 4-diones: discovery of PPAR dual {alpha}/{gamma} agonists as antidiabetic agents. Bioorg Med Chem Lett 13:2795–2798[CrossRef][Medline]
  27. Berger J, Leibowitz MD, Doebber TW, Elbrecht A, Zhang B, Zhou G, Biswas C, Cullinan CA, Hayes NS, Li Y, Tanen M, Ventre J, Wu MS, Berger GD, Mosley R, Marquis R, Santini C, Sahoo SP, Tolman RL, Smith RG, Moller DE 1999 Novel peroxisome proliferator-activated receptor (PPAR) {gamma} and PPAR{Delta} ligands produce distinct biological effects. J Biol Chem 274:6718–6725[Abstract/Free Full Text]
  28. Berger JP, Petro AE, Macnaul KL, Kelly LJ, Zhang BB, Richards K, Elbrecht A, Johnson BA, Zhou G, Doebber TW, Biswas C, Parikh M, Sharma N, Tanen MR, Thompson GM, Ventre J, Adams AD, Mosley R, Surwit RS, Moller DE 2003 Distinct properties and advantages of a novel peroxisome proliferator-activated protein [{gamma}] selective modulator. Mol Endocrinol 17:662–676[Abstract/Free Full Text]
  29. Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, Springer J 1980 Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA 77:3957–3961[Abstract/Free Full Text]
  30. Germershausen JI, Hunt VM, Bostedor RG, Bailey PJ, Karkas JD, Alberts AW 1989 Tissue selectivity of the cholesterol-lowering agents lovastatin, simvastatin and pravastatin in rats in vivo. Biochem Biophys Res Commun 158:667–675[CrossRef][Medline]
  31. Heller RA, Shrewsbury MA 1976 3-Hydroxy-3-methylglutaryl coenzyme A reductase from rat liver. Its purification, properties, and immunochemical studies. J Biol Chem 251:3815–3822[Abstract/Free Full Text]
  32. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[Free Full Text]
  33. Guo Q, Wang PR, Milot DP, Ippolito MC, Hernandez M, Burton CA, Wright SD, Chao Y 2001 Regulation of lipid metabolism and gene expression by fenofibrate in hamsters. Biochim Biophys Acta 1533:220–232[Medline]
  34. Packard CJ 1998 Overview of fenofibrate. Eur Heart J 19(Suppl A):A62–A65
  35. Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, Fruchart JC, Rubin E, Denefle P, Staels B, Branellec D 1996 Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest 97:2408–2416[Medline]
  36. Vu-Dac N, Chopin-Delannoy S, Gervois P, Bonnelye E, Martin G, Fruchart JC, Laudet V, Staels B 1998 The nuclear receptors peroxisome proliferator-activated receptor {alpha} and Rev-erb{alpha} mediate the species-specific regulation of apolipoprotein A-I expression by fibrates. J Biol Chem 273:25713–25720[Abstract/Free Full Text]
  37. Goldstein JL, Brown MS 1990 Regulation of the mevalonate pathway. Nature 343:425–430[CrossRef][Medline]
  38. Krause BR, Princen HM 1998 Lack of predictability of classical animal models for hypolipidemic activity: a good time for mice? Atherosclerosis 140:15–24[CrossRef][Medline]
  39. Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA, Briggs M, Deeb S, Staels B, Auwerx J 1996 PPAR{alpha} and PPAR{gamma} activators direct a distinct tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 15:5336–5348[Medline]
  40. Staels B, Vu-Dac N, Kosykh VA, Saladin R, Fruchart JC, Dallongeville J, Auwerx J 1995 Fibrates down-regulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the hypolipidemic action of fibrates. J Clin Invest 95:705–712
  41. Williams KJ, Petrie KA, Brocia RW, Swenson TL 1991 Lipoprotein lipase modulates net secretory output of apolipoprotein B in vitro. A possible pathophysiologic explanation for familial combined hyperlipidemia. J Clin Invest 88:1300–1306
  42. Collins R, Armitage J, Parish S, Sleigh P, Peto R 2003 MRC/BHF Heart Protection Study of cholesterol-lowering with simvastatin in 5963 people with diabetes: a randomised placebo-controlled trial. Lancet 361:2005–2016[CrossRef][Medline]
  43. Prueksaritanont T, Zhao JJ, Ma B, Roadcap BA, Tang C, Qiu Y, Liu L, Lin JH, Pearson PG, Baillie TA 2002 Mechanistic studies on metabolic interactions between gemfibrozil and statins. J Pharmacol Exp Ther 301:1042–1051[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Pharmacol. Rev.Home page
L. Michalik, J. Auwerx, J. P. Berger, V. K. Chatterjee, C. K. Glass, F. J. Gonzalez, P. A. Grimaldi, T. Kadowaki, M. A. Lazar, S. O'Rahilly, et al.
International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors
Pharmacol. Rev., December 1, 2006; 58(4): 726 - 741.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
H. Liu, C. Zang, M. H. Fenner, D. Liu, K. Possinger, H. P. Koeffler, and E. Elstner
Growth inhibition and apoptosis in human Philadelphia chromosome-positive lymphoblastic leukemia cell lines by treatment with the dual PPAR{alpha}/{gamma} ligand TZD18
Blood, May 1, 2006; 107(9): 3683 - 3692.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
P. Shen, M. H. Liu, T. Y. Ng, Y. H. Chan, and E. L. Yong
Differential Effects of Isoflavones, from Astragalus Membranaceus and Pueraria Thomsonii, on the Activation of PPAR{alpha}, PPAR{gamma}, and Adipocyte Differentiation In Vitro
J. Nutr., April 1, 2006; 136(4): 899 - 905.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/4/1640    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services