A New Thiazolidinedione, NC-2100, Which Is a Weak PPAR

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A New Thiazolidinedione, NC-2100, Which Is a Weak PPAR- Activator, Exhibits Potent Antidiabetic Effects and Induces Uncoupling Protein 1 in White Adipose Tissue of KKAy Obese Mice Yuka Fukui, Sei-ichiro Masui, Shiho Osada, Kazuhiko Umesono, and Kiyoto Motojima

Thiazolidinediones (TZDs) reduce insulin resistance in in subcutaneous WAT, although pioglitazone and trogli- type 2 diabetes by increasing peripheral uptake of glu- tazone also slightly induced UCP1 only in mesenteric cose, and they bind to and activate the transcriptional WAT. These characteristics of NC-2100 should be bene- factor peroxisome proliferator-activated receptor- ficial for humans with limited amounts of brown adipose (PPAR-). Studies have suggested that TZD-induced tissue. Diabetes 49:759–767, 2000 activation of PPAR- correlates with antidiabetic action, but the mechanism by which the activated PPAR- is involved in reducing insulin resistance is not known. To examine whether activation of PPAR- directly correlates ype 2 diabetes is characterized by increased blood with antidiabetic activities, we compared the effects of glucose levels that cannot be adequately con- 4 TZDs (troglitazone, pioglitazone, BRL-49653, and a trolled by insulin or insulin secretion–stimulating new derivative, NC-2100) on the activation of PPAR- in sulfonylureas (1,2). To prevent diabetes-induced a reporter assay, transcription of the target genes, adi- T complications such as coronary heart disease, blindness, and pogenesis, plasma glucose and triglyceride levels, and kidney failure, new drugs that reduce peripheral insulin resis- body weight using obese KKAy mice. There were 10- to tance have been developed (3,4). Thiazolidinedione (TZD) 30-fold higher concentrations of NC-2100 required for derivatives appear to increase peripheral uptake of glucose, maximal activation of PPAR- in a reporter assay system, and only high concentrations of NC-2100 weakly induced which reduces insulin resistance (3,5). However, how TZDs transcription of the PPAR- but not PPAR- target increase glucose uptake and their contribution to the reduc- genes in a whole mouse and adipogenesis of cultured tion of insulin resistance are unclear. On the other hand, TZDs 3T3L1 cells, which indicates that NC-2100 is a weak have recently been shown to directly bind to and activate a PPAR- activator. However, low concentrations of transcriptional factor, peroxisome proliferator-activated NC-2100 efficiently lowered plasma glucose levels in receptor- (PPAR-) (6–8). PPARs have unique tissue-specific KKAy obese mice. These results strongly suggest that roles in lipid homeostasis. PPAR- is expressed at high levels TZD-induced activation of PPAR- does not directly cor- in the liver (a tissue with a high capacity for -oxidation of fatty relate with antidiabetic (glucose-lowering) action. Fur- acids), and its roles include involvement in fatty acid catabo- thermore, NC-2100 caused the smallest body weight lism (9,10). PPAR- is most abundant in adipose tissues and increase of the 4 TZDs, which may be partly explained by the finding that NC-2100 efficiently induces uncoupling plays an important role in adipogenesis. The natural ligands protein (UCP)-2 mRNA and significantly induces UCP1 for PPARs have not been definitively identified, although bind- mRNA in white adipose tissue (WAT). NC-2100 induced ing of ligands is required for transcriptional activation. TZDs UCP1 efficiently in mesenteric WAT and less efficiently as well as prostaglandin J2 derivatives are artificial ligands for PPAR- and promote adipogenesis in cell cultures (6,7,11). From the Department of Biochemistry (Y.F., K.M.), School of Pharmaceu- Studies have suggested that TZD-induced activation of tical Sciences, Toho University, Chiba; the Institute for Virus Research PPAR- correlates with adipogenic activities and with antidi- (S.O., K.U.), Kyoto University, Kyoto; and the Research Laboratories (Y.F., abetic actions (12). However, these results were based on a lim- S.M.), Nippon Chemiphar Company, Saitama, Japan. ited number of TZD derivatives, and how the activated Address correspondence and reprint requests to Kiyoto Motojima, PhD, Department of Biochemistry, School of Pharmaceutical Sciences, Toho PPAR- is involved in increasing peripheral glucose uptake University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan. E-mail: moto- activities is unclear. [email protected]. In this study, we analyzed a novel TZD, NC-2100, that weakly Received for publication 31 December 1999 and accepted in revised form 24 January 2000. activates PPAR- . By comparing the effects of TZDs with vari- A-FAB, adipocyte fatty acid binding protein; apo(E), apolipoprotein(E); ous PPAR- agonist activities on the plasma glucose levels of BAT, brown adipose tissue; CTE-1, cytosolic acyl-CoA thioesterase; DMEM, KKAy obese mice (13), we examined whether TZD-induced Dulbecco’s modified Eagle’s medium; FATP, fatty acid transport protein; FCS, fetal calf serum; GPDH, glycerol-3-phosphate dehydrogenase; HD, per- activation of PPAR- directly correlates with adipogenic activ- oxisomal hydratase-dehydrogenase; H-FAB, heart muscle–type fatty acid ities and with antidiabetic action. We also examined possible binding protein; LBD, ligand binding domain; LPL, lipoprotein lipase; PCR, mechanisms to explain why some but not all TZDs do not pro- polymerase chain reaction; PPAR-, peroxisome proliferator-activated mote obesity despite their similar antidiabetic activities receptor-; RT, reverse transcription; TZD, thiazolidinedione; UCP, uncoupling protein; VLACS, very-long-chain acyl-CoA synthetase; WAT, white adipose tis- because the TZDs had diverse effects on body and fat weights, sue; WY14,643, [4-choloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]acetic acid. and some reports have shown that troglitazone, the first TZD to

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a control powder diet (MF; Oriental Kobo, Tokyo) or a diet containing 0.5% clofibrate, 0.01 or 0.03% pioglitazone, 0.1 or 0.3% troglitazone, or 0.03 or 0.1% NC-2100 for 8 or 14 days, respectively. All of the animals were killed at day 9 to extract total RNA or at day 15 for the determination of fat mass. Food intake was measured every day. Cell culture and adipocyte differentiation assay. NIH3T3L1 mouse fibroblasts (American Type Culture Collection, Rockville, MD) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1 mg/l gentamicin. The cells were seeded and grown to con-

fluence in a 24-well culture plate in a 5% CO2 atmosphere at 37°C. At postconfluence, the medium was changed to DMEM/5% FCS supplemented with 0.5 mmol/l 1-methyl-3-isobutylxanthin, 0.5 µmol/l dexamethasone, and 1 mg/l gentamicin for 48h. The medium was then treated with test chemicals in DMEM/5% FCS (23). The activities of glycerol-3-phosphate dehydrogenase (GPDH) in the cells were measured at 9 days postconfluence as described (24). Transient transfection reporter assay. CV-1 cells were tranfected with the (GAL4-UASx4)-TK-luciferase reporter vector (25), sPG-GAL4-mPPAR--ligand binding domain (LBD) or sPG-GAL4-mPPAR--LBD (26) expression vectors, or pCMX-GAL--galactosidase expression vector by liposomal delivery (1,2- dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide [Gibco BRL, Rockville, MD]). NC-2100 or typical PPAR activators were added 6 h after transfection at concentrations described in the figure legends. At 10 h after the addition of the compounds, the transfected cells were lysed, and luciferase and -galactosidase activities were determined. Results were normalized to -galactosidase activity (26). RNA preparation and Northern blot analysis. The animals were killed by decapitation. Liver, intestine, lumbar subcutaneous WAT, mesenteric WAT, and interscapular BAT were removed and were quickly frozen in liquid nitrogen. The samples were stored at –80°C until extraction of total RNA. Total RNA was prepared from the liver, intestine, adipose tissues, or the cultured NIH3T3L1 cells by the acid guanidium thiocyanate-phenol-chloroform extraction method (27). Northern blot analysis was carried out essentially as previously described (28). The cDNA probes included peroxisomal hydratase-dehydrogenase (HD) bifunctional enzyme, apolipoprotein(E) [apo(E)], apo (AIV), adipocyte fatty acid binding protein (A-FAB), fatty acid transport protein (FATP), lipoprotein lipase (LPL), and UCP2 and have been previously described (29). The cDNAs for UCP1, UCP3, cytosolic acyl-CoA thioesterase (CTE-1), very-long-chain acyl-CoA synthetase (VLACS), and ribosomal protein S14 were obtained by cloning polymerase chain reaction (PCR) products of cDNA synthesized from the livers of mice fed with [4-choloro-6-(2,3-xylidino)-2-pyrimidinyl-thio]acetic acid (WY14,643) (Tokyo- Kasei, Tokyo) as previously described (29). The synthetic oligonucleotides used to amplify respective cDNA sequences were 5-TACACGGGGACCTAC AATGCT and 5-TCGCACAGCTTGGTACGCTT for UCP1 (corresponding to nucleotide numbers from 691 to 978 of the published mouse sequence) (30) (GenBank accession number U63419), 5-TGCACCGCCAGATGAGTTTTG and 5-GGGAGCGTTCATGTATCGGG for UCP3 (corresponding to nucleotide numbers from 432 to 931 of the published mouse sequence) (31) (Gen- FIG. 1. Structures of NC-2100, troglitazone, pioglitazone, and Bank accession number AB01742), 5 -AACCTGGACCCTTTCCTGGGATCA BRL-49653. and 5 -GGCTCCCCTCCAAAGGTGATGTTG for CTE-1 (corresponding to nucleotide numbers from 466 to 1165 of the published mouse sequence) (32) (GenBank accession number Y14004), and 5-CCTACGTGTGGATCTGGCTG GGAC and 5-GGCCGTGATTCCACTCGACATAGC for VLACS (corresponding be clinically used in several countries, increases body weight to nucleotide numbers from 396 to 817 of the published mouse sequence) (33) (14,15). We therefore compared the effects of TZDs on the (GenBank accession number AJ223958). The cDNA for heart muscle–type expression of uncoupling proteins (UCPs) 1, 2, and 3 (16–18) in fatty acid binding protein (H-FAB) was obtained as described (29) using oligo-tailed cDNA, oligo, and a specific primer: 5-GATGGTAGTAGGCTTGG white adipose tissue (WAT) and brown adipose tissue (BAT). TCATGCTACCGACCTG (corresponding to nucleotide numbers from 115 to UCPs uncouple proton movement and ATP synthesis at the 147 of the published sequence) (34) (GenBank accession number MMH- inner membrane of mitochondria and play a major role in FABP). After hybridization, membranes were washed and autoradiographed energy expenditure (19). We report herein that NC-2100 and, to with an intensifying screen at –80°C. For quantitative analysis, a BAS2000 32 a lesser extent, pioglitazone effectively induce BAT-speciﬁc image analyzer (Fujix, Tokyo) was used. [- P]dCTP (3,000 Ci/mmol) and a random prime labeling kit were purchased from Muromachi Kagaku (Tokyo) UCP1 in the WAT of obese KKAy mice. and Takara (Osaka, Japan), respectively. Determination of plasma glucose and triglyceride levels. Blood samples RESEARCH DESIGN AND METHODS were collected from the tail vein. Plasma glucose and triglyceride levels were Drugs. The 2-(p-chlorophenoxy)isobutyric acid ethyl ester (clofibrate) was measured with the Glucose-CII test and the triglyceride-G test, respectively purchased from Tokyo-Kasei (Tokyo). All of the TZDs were synthesized at Nip- (Wako Pure Chemical, Osaka, Japan). pon Chemiphar (Misato, Japan). Troglitazone, pioglitazone-HCl (pioglita- Determination of fat mass. The animals were killed with ether. As much zone), and BRL-49653 were synthesized as previously described (20–22). fat tissue as possible was collected from trunk subcutaneous tissue, epi- NC-2100 {[±]-5-[(7-benzyloxy-3-quinolyl)methyl]-2,4-thiazolidinedione} was didymal tissue, mesenteric WAT, and interscapular BAT. Wet weights were synthesized using methods described in Patent Cooperation Treaty patent measured. WO-9612719. The structure of NC-2100 is given in Fig. 1. Determination of sizes of white adipocytes. Mesenteric and subcuta- Animals and treatment. Male KKAy/TaJcl obese mice were purchased from neous fat tissues were separated. Paraffin sections were prepared and stained Clea Japan (Tokyo) (13), were kept on a 12-h light–dark cycle, and were pro- with hematoxylin and eosin. The size of each adipocyte was determined with vided with food and water ad libitum. The 11-week-old mice were fed either a microscope and image analysis software.

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A domain and the reporter luciferase gene that has the receptor binding site. The effects of NC-2100 on the expression of luciferase were investigated in comparison with typical PPAR- activators (troglitazone, pioglitazone, and BRL- 49653) and PPAR- activators (5,8,11,14-eicosatetraynoic acid and Wy14,643). NC-2100 activated PPAR- in a dose- dependent manner with a maximal activation nearly equal to those of BRL-49653 and pioglitazone (Fig. 2B). However, NC-2100 was weaker than these TZDs, with a 50% effective concentration (EC50) of 10- to 30-fold greater. Unexpectedly, NC-2100 also activated PPAR- in a similar manner to PPAR- (Fig. 2A). Thus, the in vitro transient transfection reporter assay showed that NC-2100 was a weak activator for both PPAR- and PPAR-. Compounds that exhibit PPAR-– or PPAR-–activating activities in forced expression systems with CV-1 cells and chimeric genes do not always activate the corresponding receptor in vivo. Therefore, we next examined the in vivo effect of NC-2100 with whole animals and cultured cells. First, KKAy obese mice were fed a diet containing NC-2100 or a control diet containing clofibrate, troglitazone or pioglitazone, and PPAR- activators to compare their in vivo B effects. After feeding for 8 days, total RNA was isolated from the livers and WAT of mice, and the levels of several mRNAs were examined with Northern blots (Fig. 3). Among the PPAR-–regulated genes, HD bifunctional enzyme and CTE-1 (32) were activated, whereas apo(AIV) was downregulated in the liver by clofibrate as reported (35) but not by NC-2100 or control TZDs. In contrast, NC-2100 and control TZDs activated transcription of the PPAR- target genes, including A-FAB, FATP, and LPL in WAT (29). Compared with 0.1% of NC-2100 or other TZDs, 0.03% NC-2100 was clearly less effective, although 0.1% NC-2100 showed a similar antidiabetic effect. These results obtained with whole animals suggest that NC-2100 does not activate PPAR- but weakly activates PPAR-. Several groups have reported that PPAR- ligands promote adipocyte differentiation in various cultured fibroblasts and mesenchymal stem cell line systems (6,7,12). Thus, we next examined whether NC-2100 promotes the terminal differentiation of the preadipocyte cell line NIH3T3L1. As mark- ers of adipocyte differentiation, we measured increases in the activities of GPDH (24) in cells treated with various concentrations of NC-2100 or pioglitazone and troglitazone as control TZDs (Fig. 4). NC-2100 promoted terminal differentiation FIG. 2. NC-2100 activates both PPAR- and PPAR- in the transient in a dose-dependent manner, although it was less effective transfection reporter assay. A dose response of activation of GAL4- PPAR- (A) and GAL4-PPAR- (B) by NC-2100 and typical activators is than the control TZDs. BRL-49653 was the strongest inducer shown. CV-1 cells were transfected with (GAL4-UASx4)-TK-luciferase of terminal differentiation. It was about 50-fold stronger than reporter plasmid and GAL4-mPPAR--LBD or GAL4-PPAR-LBD plasmid troglitazone or pioglitazone. NC-2100 was an ~30-fold weaker and then treated with the indicated activator. Results were normalized inducer of GPDH activities than troglitazone or pioglitazone. to -galactosidase activity (pCMX--GAL). The concentrations of various TZDs required for adipocyte differentiation of cultured fibroblasts differed significantly, but Statistical analysis. Data are means ± SE. Statistical evaluation was per- these differences did not directly correspond to their antidi- formed with analysis of variance, Tukey’s multiple comparison test, and Dun- abetic activities (see below). nett’s test as appropriate. Although NC-2100 activated both PPAR- and PPAR- sim- ilarly in the in vitro system, all in vivo analyses performed RESULTS strongly suggest that NC-2100 is a weak but specific PPAR- NC-2100 is a weak activator for both PPAR- and activator, at least in whole animals. PPAR- in vitro but is a weak but specific PPAR- NC-2100 has a strong antidiabetic activity. TZDs are activator in vivo. To examine whether a new TZD, NC- known to increase the sensitivity of peripheral tissues to 2100, activates PPAR-, we first used an in vitro system. The insulin via an unclear mechanism, and their ability to activate CV-1 cell line was cotransfected with chimeric receptor plas- PPAR- alone may not be sufficient. Thus, we next examined mids that have mouse PPAR- or PPAR- ligand binding the effect of NC-2100 on plasma glucose and triglyceride lev-

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FIG. 3. NC-2100 induces mRNA of PPAR- but not PPAR- target genes in the tissues of mice. KKAy obese mice were fed either a control diet or a diet containing 0.5% clofibrate (Clofib 0.5), 0.01% pioglitazone (Piogli 0.01), 0.1% troglitazone (Trogli 0.1), 0.3% troglitazone (Trogli 0.3), and 0.1% (NC-2100 0.1) or 0.3% NC-2100 (NC-2100 0.3) for 8 days. Total RNA (5 µg) isolated from liver (A) and mesenteric WAT (B) from 5 mice in each group were pooled and analyzed by Northern blot with the cDNAs for HD, CTE-1, apo(AIV), apo(E) (RNA loading control for liver samples), A-FAB, FATP, LPL, and S14 (controls for adipose samples). els with KKAy obese mice. As shown in Fig. 5, treatment of but troglitazone did not. The differences in the effects on fat the obese mice with 0.1% NC-2100 for 1 or 2 weeks signifi- weights and on the sizes of adipocytes may partly explain a cantly lowered both glucose and triglyceride levels to levels unique feature of NC-2100 that does not induce body weight comparable with 0.03% pioglitazone treatment and to levels increase as other TZDs do. lower than with 0.1% troglitazone treatment. NC-2100 and NC-2100 induces ectopic expression of UCP1 in WAT. pioglitazone decreased free fatty acid levels to nearly 50% of Our results showing that NC-2100 induced large increases in the control level, whereas troglitazone had little effect food intake but not in body weight suggest that energy expen- (unpublished data). These data indicate that NC-2100, even diture was stimulated. UCP1 in BAT contributes to energy at 0.1%, has antidiabetic activities comparable with troglita- expenditure (16). Recent studies have identified 2 additional zone and pioglitazone. proteins, UCP2 (17) and UCP3 (18), with sequence homologies NC-2100 has a small effect on body weight and fat to and similar functional properties as UCP1. Therefore, we weight. In our study, NC-2100 caused the smallest increases measured the changes in the levels of the mRNA for UCP1, in body weight and fat mass (Fig. 6). Pioglitazone induced an UCP2, and UCP3 in several tissues of mice treated with ~20% increase in body weight and at least a 2-fold increase NC-2100 and control drugs. Representative Northern blots in the weight of subcutaneous WAT and BAT over 2 weeks. for UCP1, UCP2, and UCP3 are shown in Fig. 8. UCP2 mRNA Troglitazone induced an ~10% increase in body weight with levels increased only in subcutaneous WAT in response to a small effect on fat weight. In contrast, NC-2100 induced at clofibrate and TZDs. Among these, NC-2100 was the most maximum a 5% increase in body weight with no effect on the effective. In other tissues, including BAT (36), we did not weight of mesenteric WAT deposits. NC-2100 apparently detect significant increases in the mRNA. UCP1 mRNA increased the weight of the subcutaneous WAT deposits expression increased in BAT in response to all TZDs as pre- more than troglitazone (Fig. 6B), but the difference between viously reported (37), but the extent of induction was small. NC-2100 and troglitazone was not statistically significant. Above all, interestingly, UCP1 mRNA was induced both in Similarities regarding the effects of NC-2100 and troglita- mesenteric and subcutaneous WAT by NC-2100 in a dose- zone on the weight of the subcutaneous WAT deposits as well dependent manner. The levels of mRNA induced by 0.1% as a strong fat weight–increasing activity of pioglitazone NC-2100 were nearly 10% in subcutaneous WAT and 0.5–1.0% were confirmed by measuring the sizes of adipocytes in the in mesenteric WAT of the normal level of UCP1 mRNA in fat tissues (Fig. 7). These differences do not simply reflect BAT. Pioglitazone and troglitazone also induced UCP1 only in changes in food intake because pioglitazone and NC-2100 mesenteric WAT, and the levels were quite low. BAT contam- stimulated food intake by 40–50% and 20–30%, respectively, ination during tissue separation and the conversion of some

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white adipocytes to typical brown adipocytes were ruled out by screening for H-FAB, which is exclusive to BAT (38) (Fig. 8B). Another BAT-predominant mRNA for VLACS (Y.F., K.M., unpublished data) that may be a possible fatty acid transporter instead of a peroxisomal acetyl-CoA synthetase (39) also was not induced in these WATs by NC-2100. Piogli- tazone and troglitazone caused weak UCP1 mRNA induction in mesenteric WAT and negligible induction in subcutaneous WAT when compared with NC-2100. Finally, we examined whether NC-2100 induces UCP1 in 3T3L1 cells. NC-2100 and other TZDs, including BRL-49653, did not induce UCP1 mRNA in differentiating or differentiated 3T3L1 cells, although all induced A-FAB (data not shown). Thus, under the conditions in this study, TZDs can induce BAT- speciﬁc UCP1 in the WAT of obese mice but not in vitro.

DISCUSSION TZDs such as troglitazone, pioglitazone, and BRL-49653 improve insulin resistance by enhancing insulin action in FIG. 4. NC-2100 induces differentiation of NIH3T3L1 cells to skeletal muscle, liver, and adipose tissues (3,5) through a adipocytes. NIH3T3L1 cells were treated with increasing concentra- mechanism that is not yet fully understood. TZDs are ligands tions of NC-2100, troglitazone, pioglitazone, or BRL-49653 for 7 days, and GPDH activities were subsequently measured with total cell extracts. Data are means ± SE for 6 or 7 determinations. A

B B

FIG. 6. Effects of 3 TZDs on body and adipose tissue weights. Male KKAy obese mice received test compounds for 14 days as described in Fig. 5. Body weights were measured every other day (A). At day 15, the FIG. 5. NC-2100 lowers plasma glucose and triglyceride levels in KKAy animals were killed, and several adipose tissues were separated for obese mice. Male KKAy obese mice received either a control diet or a weight determination (B). Data are means ± SE for 7 animals per treat- diet containing 0.1% NC-2100, 0.03% pioglitazone, or 0.1% troglitazone ment group. Differences between control and treatment groups were for 14 days. Blood samples were collected, and plasma glucose (A) and statistically evaluated by analysis of variance followed by Tukey’s plasma triglyceride (B) levels were measured. Data are means ± SE for multiple comparison test for body weight and Dunnett’s test for adi- 7 animals per treatment group. Differences between control and pose tissue weight. , Control; , NC-2100 (0.1%); , pioglitazone HCl treatment groups were statistically evaluated by Dunnett’s test. *P < (0.03%); , troglitazone (0.1%). *P < 0.05 vs. control; #P < 0.01 vs. 0.05 vs. control; #P < 0.01 vs. control. control.

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FIG. 7. Effects of 3 TZDs on the size of white adipocytes. Male KKAy obese mice received test compounds for 14 days as described in Fig. 5. At day 15, the animals were killed, and mesenteric (A) and subcutaneous (B) fat tissues were separated. Paraffin sections were prepared from each sample and were stained by hematoxylin and eosin. The sizes of adipocytes were determined with a microscope and image analysis software. Data are means ± SE for 7 animals per treatment group. Differences between control and treatment groups were statistically evaluated with Dunnett’s test. NC-2100 0.1, NC-2100 0.1%; Piogli 0.03, pioglitazone 0.03%; Trogli 0.1, troglitazone 0.1%. *P < 0.05. for PPAR- (6–8), and studies have suggested that TZD- required to induce differentiation of preadipose cells to induced activation of PPAR- correlates with antidiabetic adipocytes (25). However, doses of NC-2100 (0.1%) similar to action (12). A new TZD, NC-2100, weakly activated PPAR- other TZDs had similar or better antidiabetic effects in obese (Fig. 2B) and induced the expression of PPAR- target genes mice (Fig. 5). Our preliminary examination on the half-lives and (Fig. 3) and adipocyte differentiation (Fig. 4). It also exerted bioavailabilities of these TZDs in murine bodies suggests that PPAR-–activating activities only in the transient transfec- the differences in these factors alone cannot explain the dis- tion reporter assay (Fig. 2). All of the compounds that exhibit crepancies between adipogenesis induction and antidiabetic PPAR-– or PPAR-–activating activities in this artificial and effects. We hypothesize that the ability to activate PPAR- is forced expression system do not activate the corresponding necessary but not sufficient for antidiabetic activity of TZDs, receptor in vivo, and we conclude that NC-2100 is a weak but and TZDs have other in vivo targets that are crucial for full specific PPAR- activator based on the results from in vivo antidiabetic action. Recent studies showing that TZDs have var- studies. The requirement for high concentrations of NC-2100 ious effects may strengthen our claim (42,43). More recently, to activate PPAR- in the transient transfection reporter assay another new TZD, MCC-555, was reported to be a potent and to induce adipogenesis can be explained by its low affin- antidiabetic compound with low activities to induce adipocyte ity to PPAR-. In addition to the conserved head group that differentiation and a low affinity for PPAR- (44), although interacts specifically with the receptor and a divergent whether MCC-555 induces UCP1 in WAT is not known. hydrophobic part that interacts with the large ligand-binding The 4 TZDs also exerted different effects on food intake, pocket of the receptor in a relatively nonspecific manner as fat mass increases, plasma fatty acid levels, and target gene suggested by a recent X-ray structure analysis (40), NC-2100 activation, which further suggests that TZDs interact with has a unique nitrogen in the quinoline nucleus (Fig. 1). This more than just PPAR-. However, in contrast with the strict basic nitrogen in the center part lies in an -helix (41) and may ligand specificity of the nuclear hormone receptors, PPARs interfere with receptor interaction, thus significantly reducing bind and are activated by structurally diverse natural and affinity compared with other TZDs. artificial compounds, and binding of ligands with diverse To examine whether the potencies of the compounds to structures may cause various conformational changes in the activate PPAR- correlate with their antidiabetic activities receptor, thus leading to various transcriptional complex for- (12), we compared 4 TZDs in vivo and in cultured differentiated mations that contain different factors such as coactivators. cell systems and found that the suggested correlation was not Thus, the repertoire of the target genes of 1 type of PPAR may entirely confirmed. First, we found that the concentrations of depend on the structures of the ligands. Some artificial each TZD required for adipocyte differentiation of NIH3T3L1 ligands (drugs) may limit the target genes or cause an exag- cells were quite different (Fig. 4). Ectopic expression of gerated response of 1 gene. PPAR- alone induced differentiation (41), and the concen- Among the different effects that TZDs exert, induction of trations of several PPAR- ligands required to activate the UCP1 in WAT by NC-2100 is noteworthy (Fig. 8). UCP1 is receptor are reported to be in good agreement with those known to be expressed only in BAT. The mouse gene has a

764 DIABETES, VOL. 49, MAY 2000 Y. FUKUI AND ASSOCIATES

FIG. 8. NC-2100 induced UCP1 but not H-FAB and VLACS mRNA in WAT. Male KKAy mice received test compounds as described in Fig. 2 (A). Total RNA (5 µg) isolated from mesenteric and subcutaneous WAT was analyzed by Northern blot with the cDNA for UCP1, UCP2, UCP3, and S14 (RNA loading control) (B). Total RNA (5 µg) isolated from interscapular BAT, subcutaneous tissue, and mesenteric WAT was analyzed by Northern blot with cDNA for UCP1, H-FAB, and VLACS. Clofib 0.5, clofibrate 0.5%; NC-2100, 0.1, NC-2100 0.1%; Piogli 0.01, pioglitazone 0.01%; Trogli 0.3, troglitazone 0.3%.

functional PPAR- response element in the promoter region, BAT-speciﬁc or -predominant mRNA for H-FAB (38) and a and the expression is strictly regulated in a differentiation- possible fatty acid transporter, FATP2 or VLACS (39) (Y.F., dependent manner (45). The possibility that treatment with K.M., unpublished data). Furthermore, the amounts of NC-2100 induces differentiation of brown adipocytes in WAT mRNA for PPAR- coactivator or PPAR- coactivator 1 (47) (46) is unlikely because we detected UCP1 but not other were too low to be detected by our Northern blot analysis

DIABETES, VOL. 49, MAY 2000 765 A NEW TZD INDUCES UCP1 IN WAT from the WAT of NC-2100 fed mice (data not shown). Instead, 9. Lee SS, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero some perturbation or interaction between PPAR- and other PM, Westphal H, Gonzalez FJ: Targeted disruption of the alpha isoform of the BAT-specific transcriptional factors may be induced by NC- peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15:3012– 2100 in vivo but not in the model cell system we used. To our 3022, 1995 knowledge, the first report showed that a 3-adrenergic ago- 10. Ren B, Thelen AP, Peters JM, Gonzalez FJ, Jump DB: Polyunsaturated fatty nist induced similar ectopic expression of UCP1 mRNA in acid suppression of hepatic fatty acid synthase and S14 gene expression does WAT and muscle with the reverse transcription (RT)-PCR not require peroxisome proliferator-activated receptor . J Biol Chem 272: method (48), but another report was published showing that 26827–26832, 1997 11. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand the mRNA that responded to hormones and 3-adrenergic ago- P, Wahli W, Willson TM, Lenhard JM, Lehmann JM: Fatty acids and eico- nists was UCP3 when using Northern blots (18). Detection of sanoids regulate gene expression through direct interactions with peroxisome UCP3 mRNA by Northern blot does not exclude the possibility proliferator-activated receptors alpha and gamma. Proc Natl Acad Sci U S A that UCP1 mRNA was also induced in WAT by hormones 94:4318–4328, 1997 12. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkson WO, Willson TM, Kliewer and 3-adrenergic agonists, but more quantitative analysis SA: An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome than RT-PCR will be necessary to confirm their interesting proliferator-activated receptor (PPAR). J Biol Chem 270:12953–12956, 1995 finding because we isolated UCP1 cDNA (confirmed by 13. Saha AK, Kurowski TG, Colca JR, Ruderman NB: Lipid abnormalities in tis- sequencing) with RT-PCR using poly(A) RNA from the sues of the KKAy mouse: effects of pioglitazone on malonyl-CoA and diacyl- glycerol. Am J Physiol 267:E95–E101, 1994 mouse liver as described in RESEARCH DESIGN AND METHODS. 14. Shimizu H, Tsuchiya T, Sato N, Shimomura Y, Kobayashi I, Mori M: Troglita- TZDs have been reported to induce UCP2 in rodent BAT zone reduces plasma leptin concentration but increases hunger in NIDDM (37), although we could not detect a significant increase of the patients. 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