Troglitazone Not Only Increases GLUT4 but Also Induces Its Translocation in Rat Adipocytes Mitsuyo Shintani,1 Haruo Nishimura,2 Shin Yonemitsu,1 Yoshihiro Ogawa,1 Tatsuya Hayashi,1 Kiminori Hosoda,1 Gen Inoue,1 and Kazuwa Nakao1

Thiazolidinediones, insulin-sensitizing agents, have 2 diabetes (3–5). Several mechanisms by which these been reported to increase glucose uptake along with the compounds enhance insulin action have been proposed. expression of glucose transporters in adipocytes and Under in vivo conditions, it has been suggested that cardiomyocytes. Recently, we have further suggested thiazolidinediones improve insulin resistance by enhanc- that the translocation of GLUT4 is stimulated by thia- ing adipocyte differentiation and increasing the number of zolidinediones in L6 myocytes. However, the direct small adipocytes in obese Zucker rats (6) as well as effects of thiazolidinediones on translocation of glucose correcting the deficit in glucose transporters in KKAy transporters have not yet been determined. In this diabetic mice (7). Young et al. (8) reported that repeat study, using hemagglutinin epitope-tagged GLUT4 treatment of thiazolidinedione enhances insulin action in (GLUT4-HA), we provide direct evidence of the effect of troglitazone on the translocation of GLUT4 in rat epi- white adipocytes by increasing insulin binding and cell- didymal adipocytes. Primary cultures of rat adipocytes surface GLUT4. In in vitro studies, it has been reported were transiently transfected with GLUT4-HA and over- that thiazolidinediones increased glucose uptake in 3T3- expressed eightfold compared with endogenous GLUT4 F442A and 3T3-L1 adipocytes by increasing GLUT4 and/or in transfected cells. A total of 24 h of treatment with GLUT1 content (9,10). In mature adipocytes, troglitazone troglitazone (10؊4 mol/l) increased the cell surface has also been reported to increase GLUT4 mRNA levels level of GLUT4-HA by 1.5 ؎ 0.03–fold (P < 0.01) without (11). Furthermore, thiazolidinediones have been suggested changing the total amount of GLUT4-HA, whereas it to modulate intrinsic activity and translocation of glucose increased the protein level of endogenous GLUT4 (1.4- transporters (12). Recently, we have further suggested that fold) without changing that of GLUT1. Thus, the direct the translocation of GLUT4 is stimulated by thiazo- effect on the translocation can be detected apart from the increase in endogenous GLUT4 content using lidinediones in L6 myocytes (13). However, the direct GLUT4-HA. Troglitazone not only increased the trans- effects of thiazolidinediones on the translocation of glu- location of GLUT4-HA on the cell surface in the basal cose transporters have not yet been verified. Because the state but also caused a leftward shift in the dose- total cellular GLUT4 content was increased by troglitazone response relations between GLUT4-HA translocation in adipocytes, it was difficult to determine the direct effect and insulin concentration in the medium (ED50: from of troglitazone on the translocation of GLUT4 by the ϳ0.1 to 0.03 nmol/l). These effects may partly contrib- membrane fractionation method. ute to the antidiabetic activity of troglitazone in pa- In the present study, to investigate the direct effect of tients with obesity and type 2 diabetes. Diabetes 50: troglitazone on translocation of GLUT4, we transfected 2296–2300, 2001 hemagglutinin epitope-tagged GLUT4 (GLUT4-HA) into rat adipocytes, incubated the cells in the presence or absence of troglitazone for 24 h, and detected the cell-surface GLUT4-HA. This system was chosen because troglitazone hiazolidinediones, insulin-sensitizing agents, im- did not change the expression of GLUT4-HA inserted into prove hyperglycemia and hyperinsulinemia by the expression plasmid. Moreover, it is possible to exam- increasing insulin responsiveness and/or sensi- ine the direct effect of GLUT4-HA on translocation in Ttivity in obese, type 2 diabetic, and glucose- physiologically relevant cells. We directly demonstrated intolerant patients (1,2). These compounds increase for the first time that troglitazone increases the transloca- peripheral glucose uptake, while decreasing the serum tion of GLUT4 in adipocytes. insulin level and gluconeogenesis in rodent models of type

RESEARCH DESIGN AND METHODS From the 1Department of Medicine and Clinical Science, Kyoto University Materials. Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Graduate School of Medicine, Kyoto, Japan, and the 2Department of Diabetes GIBCO/BRL (Gaithersburg, MD). Troglitazone was a generous gift from and Endocrinology, Osaka Saiseikai Nakatsu Hospital, Osaka, Japan. Sankyo (Tokyo), and all other chemicals were obtained from Sigma Chemical Address correspondence and reprint requests to Haruo Nishimura, MD, (St. Louis, MO) unless otherwise noted. PhD, Department of Diabetes and Endocrinology, Osaka Saiseikai Nakatsu Animals and adipose cell preparation. Male Sprague-Dawley rats (180–200 Hospital, 2-10-39 Shibata, Kita-ku, Osaka, Japan. E-mail: [email protected] g; Shimizu Breeding Laboratories, Kyoto, Japan) fed ad libitum were used u.ac.jp. throughout the study. The rats were housed under controlled temperature Received for publication 21 December 2000 and accepted in revised form 27 June 2001. (25°C) and lighting (12-h light-dark, lights on at 8:00 A.M.) conditions. The rats BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; were anesthetized with a gas mixture of 70% CO2 and 30% O2 and decapitated. GLUT4-HA, hemagglutinin epitope-tagged GLUT4; HA, hemagglutinin epitope; All experiments were approved by the Institutional Animal Care and Use KRBH, Krebs-Ringer bicarbonate HEPES buffer. Committee of Kyoto University. The epididymal fat pads were removed,

2296 DIABETES, VOL. 50, OCTOBER 2001 M. SHINTANI AND ASSOCIATES

minced, and digested with collagenase (type 1; Cooper Biochemical, Freehold, NJ). The preparation of the cells was carried out at 37°C in a Krebs-Ringer

bicarbonate HEPES buffer (KRBH), pH 7.4, containing 10 mmol/l NaHCO3,30 mmol/l HEPES, 1% (w/v) bovine serum albumin (BSA) (fraction V; Intergen, Purchase, NY) and 200 nmol/l adenosine as previously described (14). The cells were then washed three times with KRBH and twice with DMEM supplemented with 5% (w/v) BSA, 200 nmol/l N6-(phenyl isopropyl)-adenosine, and 75 ␮g/ml gentamicin. All incubations were carried out at 37°C in DMEM at a cytocrit of ϳ20% containing the indicated reagents. Troglitazone was dissolved in DMSO, and all cells were exposed to a final concentration of DMSO of 0.1%. Plasmid constructs. pCIS2 is an expression vector that contains a cytomeg- alovirus promoter and enhancer with a genetic intron located upstream from a multiple cloning site (obtained from C. Gorman) (15). GLUT4-HA was a pCIS2 vector containing the cDNA coding for human GLUT4 with the influenza virus hemagglutinin epitope (HA)-1 inserted in the first exofacial loop of human GLUT4. These vectors were kindly supplied by Drs. M.J. Quon FIG. 1. A: Standard curve of the blots using anti–Glut4 antibody. B: and S.W. Cushman. Expression of GLUT4-HA in rat adipocytes. Rat adipocytes were trans- Transfection of rat adipocytes. Isolated rat adipocytes were washed twice fected with the GLUT4-HA expression vector pCIS2/GLUT4-HA or ␮ with DMEM and resuspended in a cytocrit of 40%. A total of 200 ␮l of the cell pCIS2 (0.5 g/cuvette) and cultured for 24 h. Total cell lysates containing 6 ␮g total protein were subjected to SDS-PAGE and immu- suspension was added to 200 ␮l of the DMEM containing 100 ␮g of carrier noblotted with antibodies against the HA-epitope and COOH-terminus DNA (sheared herring sperm DNA; Boeringer Manheim, Mannheim, Germany) of GLUT4. The panels show representative blots from three experi- and 0.5 ␮g of the expression plasmids. Electroporation was performed in ments. 0.4-cm gap cuvettes (Bio-Rad Laboratories, Richmond, CA) with a double electric shock (800 V, 25 ␮F:200 V, and 1,050 ␮F) using an Easyject electroporator (Equibio, Kent, U.K.). After the electroporation, the cells were 1B, left lane). In the pCIS2/GLUT4-HA–transfected cells washed once in DMEM, pooled in groups of 4–10 cuvettes, and cultured in (Fig. 1B, right lane), GLUT4-HA was detected. DMEM containing 3.5% BSA with or without troglitazone (10Ϫ4 mol/l) for To quantify the relative amount of GLUT4-HA compared 16–24hat37°C in a humidified atmosphere of 5% CO2. with endogenous GLUT4 in the adipocytes, total cell Assay for cell-surface GLUT4-HA. After 16–24 h incubation, the transfected lysates were analyzed with a Western blot using a poly- adipocytes were washed in a KRBH buffer containing 5% BSA. Samples corresponding to the cells from one cuvette were distributed into 1.5-ml clonal antibody against the COOH-terminus of GLUT4. The microcentrifuge tubes. After incubation with 60 nmol/l of insulin for 30 min at antibody recognizes both endogenous GLUT4 and GLUT4- 37°C, KCN (potassium cyanide) (final concentration, 2 mmol/l) was added to HA. In pCIS2/GLUT4-HA–transfected cells, electrophoretic prevent GLUT4 redistribution. Then, cell-surface GLUT4-HA was detected mobility shift was detected in the blot using anti-GLUT4 using a monoclonal anti-HA antibody conjugated with 125I-labeled sheep anti–mouse IgG (Amersham Pharmacia Biotech). The transfection efficiency antibody because of the difference in molecular weight was ϳ5%, as estimated by immunofluorescense. The efficiency was compara- between GLUT4 and GLUT4-HA. The total amount of ble with previous studies (16–18). Cells transfected with empty vector pCIS2 GLUT4 increased 1.4-fold in the GLUT4-HA–transfected were used to determine nonspecific binding of the antibodies. The lipid weight adipocytes compared with the pCIS2-transfected cells from a 200-␮l aliquot of cells was determined as described (19) and used to (Fig. 1, lower panel). Considering that the transfection normalize the data for each sample. Immunoblotting. Total cell lysates were prepared from the adipocytes efficiency estimated by immunofluorescence study was 5% incubated with or without troglitazone (10Ϫ4 mol/l) for 24 h in DMEM. After (see RESEARCH DESIGN AND METHODS), the transfection resulted the protein concentration had been determined, dithiothreitol was added to a in an eightfold overexpression ([1.4–1.0]/0.05) of final concentration of 100 mmol/l. The proteins were separated by SDS-PAGE GLUT4-HA compared with the endogenous GLUT4 level and transferred to polyvinylidene fluoride microporous membranes (New England Nuclear Life Science Products, Boston, MA). Western blotting was per transfected cell. performed using a polyclonal antibody raised against the COOH-termini of Effect of troglitazone on translocation of GLUT4-HA. GLUT4 (14), GLUT1 (14) (kind gifts from Dr. S.W. Cushman), or a monoclonal Next, we studied the dose-response relation between anti-HA antibody (HA 11; Berkeley Antibody, Richmond, CA). Bound antibod- troglitazone and the cell surface level of GLUT4-HA in ies were visualized with secondary antibodies conjugated to horseradish adipocytes. Transfected adipocytes were incubated with peroxidase using enhanced chemiluminescence, as described by the manu- facturer (Amersham International, Buckinghamshire, U.K.), and autoradiog- or without troglitazone for 24 h, and the cell surface level Ϫ4 raphy film (Konica, Tokyo). The densities of bands on the film were measured of GLUT4-HA was determined. Troglitazone (0–10 using a scanning densitometer. To verify a quantitation of blots, we made mol/l) increased the cell-surface GLUT4-HA in a dose- standard curves of the blots using anti-GLUT4 (Fig. 1A), -HA, and -GLUT1 dependent manner (Fig. 2A). The level was increased by antibodies, respectively. Ϯ Ϫ4 Statistical analysis. Results are expressed as means Ϯ SE. Statistical 1.5-fold (148 8%) at the maximal concentration (10 significance was tested with one-way analysis of variance followed by mol/l) of troglitazone. To determine the time course, Duncan’s multiple range test or Wilcoxon’s signed-rank test, as appropriate. troglitazone was added to the medium of transfected P Ͻ 0.05 was considered significant. adipocytes 6, 12, 18, or 24 h before the detection of cell-surface GLUT4-HA (Fig. 2B inset). The cell-surface RESULTS level of GLUT4-HA was increased by troglitazone in a Expression of GLUT4-HA in rat adipocytes. To exam- time-dependent manner (Fig. 2B). ine the effect of troglitazone on the translocation of To confirm that troglitazone did not increase the expres- GLUT4, we transfected GLUT4-HA as a reporter gene in rat sion of GLUT4-HA, we evaluated the GLUT4-HA levels adipocytes and directly determined the effect on the with a Western blot using whole-cell lysates treated with distribution of GLUT4-HA. The adipocytes were trans- or without troglitazone for 24 h. The results of this fected with pCIS2/GLUT4-HA or pCIS2 (empty vector) (0.5 experiment demonstrated that there was no detectable ␮g/cuvette) and incubated for 24 h. Then, the total cell change in the GLUT4-HA levels caused by troglitazone lysates were subjected to SDS-PAGE and Western blot (Fig. 3). using an anti-HA antibody. There was no cross-reactivity Effect of troglitazone on insulin-stimulated translo- with the anti-HA antibody in the control cells (pCIS2) (Fig. cation of GLUT4-HA. We examined the effect of trogli-

DIABETES, VOL. 50, OCTOBER 2001 2297 TROGLITAZONE INCREASES TRANSLOCATION OF GLUT4

FIG. 2. Effect of troglitazone on translocation of GLUT4-HA. A: Rat adipocytes were transfected with pCIS2/GLUT4-HA (0.5 ␮g/cuvette) and incubated with various concentrations of troglitazone (0–10؊4 mol/l) for 24 h. The cell-surface levels of GLUT4-HA were detected as described in RESEARCH DESIGN AND METHODS. The cell surface–associated radioactivity was normalized to the lipid weight of the cells. B: GLUT4-HA–transfected cells were incubated with troglitazone (10؊4 mol/l) for various time courses (0–24 h). After 24 h, the cell-surface levels of GLUT4-HA were determined. The cell surface–associated radioactivity was normalized to the lipid weight of the cells. In both A and B, specific counts (GLUT4-HA) observed for the basal samples are 1,570 ؎ 178 cpm and nonspecific counts (pCIS2) are 398 ؎ 32 cpm. Results are the means ؎ SE of five independent experiments in triplicate. tazone on insulin-stimulated translocation of GLUT4-HA. further incubated with insulin (0–60 nmol/l) for 20 min, After being incubated with or without troglitazone (10Ϫ4 and the cell-surface GLUT4-HA was detected. The cell- mol/l) for 24 h, the GLUT4-HA–transfected cells were surface GLUT4-HA was increased by insulin in a dose- dependent manner. The ED50 value of the response was ϳ0.1 nmol/l, which was similar to that of insulin-stimu- lated glucose uptake (14). The insulin dose-response curve for cell-surface levels of GLUT4-HA was shifted to the left by troglitazone treatment (ED50, 0.03 nmol/l). The levels of cell-surface GLUT4-HA at the maximal concentration of insulin (60 nmol/l) were unchanged by troglitazone (Fig. 4). Effects of troglitazone on protein levels of GLUT4 and GLUT1 in rat adipocytes. We detected the protein levels of GLUT4 and GLUT1 in adipocytes after 24 h of treatment with troglitazone using Western blot analysis. As shown in Fig. 5, the protein level of GLUT4 was increased (138 Ϯ 14% of the control) (P Ͻ 0.05) by troglitazone. In contrast, troglitazone did not change the protein level of GLUT1 (104 Ϯ 5% of the controls) (Fig. 5).

DISCUSSION The purpose of the present study was to clarify the mechanisms underlying the antidiabetic effects of troglita- zone using primary cultured rat adipocytes. The results comprise the first evidence that troglitazone enhances the translocation of GLUT4 in addition to increasing the amount of GLUT4 in rat adipocytes. These effects of troglitazone on adipocytes may contribute to the anti- FIG. 3. Effect of troglitazone on GLUT4-HA content. Rat adipocytes diabetic action of this compound. were transfected with pCIS2 or pCIS2/GLUT4-HA (0.5 ␮g/cuvette) and It was difficult to determine the direct effect of troglita- incubated with troglitazone (0–10؊4 mol/l) for 24 h. Total cell lysates zone on the translocation of GLUT4 using the membrane containing 6 ␮g of the total protein were subjected to SDS-PAGE, followed by immunoblotting with an antibody against the HA-epitope. fractionation method when the endogenous GLUT4 con- The panels show representative blots from three experiments. tent was changed. In this study, to investigate the direct

2298 DIABETES, VOL. 50, OCTOBER 2001 M. SHINTANI AND ASSOCIATES

FIG. 4. Effect of insulin on the troglitazone-induced increase in translocation of GLUT4-HA. GLUT4-HA–transfected cells were incubated with or (without troglitazone (10؊4 mol/l) for 24 h. After washing, the cells were further incubated with various concentrations of insulin (0–60 nmol/l for 20 min. Then, the cell-surface levels of GLUT4-HA were determined using an antibody binding assay as described in RESEARCH DESIGN AND METHODS. The cell surface–associated radioactivity was normalized to the lipid weight of the cells. The values are the means ؎ SE of three independent experiments in triplicate. Specific counts (GLUT4-HA) observed for the basal samples are 1,600 ؎ 203 cpm and nonspecific counts F ؎ (pCIS2) are 407 52 cpm. Control cells ( ) were able to recruit GLUT4-HA to the cell surface in an insulin dose-dependent manner (ED50 of 0.1 nmol/l). The insulin dose-response curve for cells treated with troglitazone (E) was significantly different from that of the control cells. *P < 0.05 comparing troglitazone-treated cells with their respective controls. effect of troglitazone on translocation of GLUT4, apart fold (Fig. 5) but did not change the expression of from the amount of GLUT4, we transfected GLUT4-HA GLUT4-HA in this transfection system. The ratio of into rat adipocytes. One advantage of our system was the GLUT4-HA to total GLUT4 (GLUT4-HA ϩ endogenous ability to use GLUT4-HA as a reporter gene. Expression of GLUT4) in the adipocytes treated with troglitazone was GLUT4-HA was ϳ5% of the adipocytes subjected to elec- changed by only 4% compared with those without trogli- troporation. This system allowed us to overcome the tazone treatment (from 8/[8 ϩ 1] to 8/[8 ϩ 1.4]). When we potential difficulty of studying such a small population of determined the direct effect on translocation of GLUT4- cells using GLUT4-HA as a reporter gene (16). Therefore, HA, the troglitazone-induced increase in endogenous we could study transiently transfected cells exclusively GLUT4 (ϳ140%) was overcome by 800% overexpression of without interference from nontransfected cells. Another GLUT4-HA. Thus, it was possible to examine the direct advantage is that 24 h of incubation decreases the endog- effect on translocation using GLUT4-HA as a reporter enous GLUT4 content in rat adipocytes (20). Under this when endogenous GLUT4 increased. condition, GLUT4-HA is overexpressed compared with The cell-surface level of GLUT4-HA was increased by endogenous GLUT4. The decrease in endogenous GLUT4 troglitazone in a time- and dose-dependent manner (Fig. increases the ratio of GLUT4-HA to total GLUT4 content. 2). Troglitazone (10Ϫ4 mol/l) caused a leftward shift of the GLUT4-HA was overexpressed by eightfold compared dose-response curve between insulin and translocation of with endogenous GLUT4 in the transfected adipocytes. GLUT4-HA, without changing the maximal response (Fig. Troglitazone increased endogenous GLUT4 levels by 1.4- 4). Thus, troglitazone increases the insulin sensitivity of GLUT4 translocation in adipocytes. This suggested that the pathway of the signaling by troglitazone converges with that activated by insulin. We have recently demon- strated that troglitazone also increases translocation of GLUT4 in L6 myotubes (13). In L6 myotubes, it is supposed that troglitazone acts at a step beyond PI3K and PKB in which GLUT4 endocytosis is enhanced or reduces endo- cytosis of GLUT4. Further study is necessary to clarify the FIG. 5. Effect of troglitazone on protein levels of GLUT4 and GLUT1 in precise site of action of troglitazone with regard to the rat adipocytes. Adipocytes were preincubated with or without trogli- exocytosis and endocytosis of GLUT4. tazone (10؊4 mol/l) in DMEM for 24 h. After incubation for 24 h, total cell lysates containing 6 or 15 ␮g total protein were subjected to Long-term incubation was required for troglitazone to SDS-PAGE and immunoblotted with antibodies against the COOH- significantly increase the translocation of GLUT4 in adipo- terminus of GLUT4 or GLUT1, respectively. The panels show represen- cytes. In our recent study (13), inhibition of protein tative blots from three experiments. Densitometric scanning was used to determine the relative amount of GLUT4 protein. Ⅺ, control; f, synthesis by cycloheximide completely eliminated the troglitazone. *P < 0.05 vs. control. increase in translocation of GLUT4 by troglitazone in L6

DIABETES, VOL. 50, OCTOBER 2001 2299 TROGLITAZONE INCREASES TRANSLOCATION OF GLUT4 myotubes. It is tempting to speculate that troglitazone D, McPherson RK, Hall KL, Danbury BH, Gibbs M, Kreutter D: Actions of affects the synthesis of some molecules involved in the novel antidiabetic agent englitazone in hyperglycemic hyperinsulinemic ob/ob mice. Diabetes 39:1218–1227, 1990 translocation of GLUT4. We have detected a protein of 90 5. Oakes ND, Kennedy CJ, Jenkins AB, Laybutt DR, Chisholm DJ, Kraegen kDa associated with GLUT4-containing vesicles in adipo- EW: A new antidiabetic agent, BRL 49653, reduces lipid availability and cytes by SDS-PAGE, which was increased by troglitazone improves insulin action and glucoregulation in the rat. Diabetes 43:1203– treatment (unpublished data). It has recently been re- 1210, 1994 ported that annexin II, which may be involved in vesicular 6. Okuno A, Tamemoto H, Tobe K, Ueki K, Mori Y, Iwamoto K, Umesono K, transport, is increased by troglitazone treatment after 24 h Akanuma Y, Fujiwara T, Horikoshi H, Yazaki Y, Kadowaki T: Troglitazone increases the number of small adipocytes without the change of white in 3T3-L1 adipocytes (21). These molecules may be in- adipose tissue mass in obese Zucker rats. J Clin Invest 101:1354–1361, volved in the increase in translocation of GLUT4. 1998 Under in vivo conditions, it has been suggested that 7. Hofmann C, Lorenz K, Colca JR: Glucose transport deficiency in diabetic thiazolidinediones improve insulin resistance by enhanc- animals is corrected by treatment with the oral antihyperglycemic agent ing adipocyte differentiation and increasing the number of piogllitazone. Endocrinology 129:1915–1925, 1991 8. Young PW, Cawthorne MA, Coyle PJ, Holder JC, Holman GD, Kozka IJ, small adipocytes in obese Zucker rats (6) and correct the Kirkham DM, Lister CA, Smith SA: Repeat treatment of obese mice with deficit in glucose transporters in KKAy diabetic mice (7). BRL 49653, a new and potent insulin sensitizer, enhances insulin action in In in vitro studies, it has been reported that thiazo- white adiocytes: association with increased insulin binding and cell- lidinediones increase glucose uptake in 3T3-F442A and surface GLUT4 as measured by photoaffinity labeling. Diabetes 44:1087– 1092, 1995 3T3-L1 adipocytes by increasing GLUT4 and/or GLUT1 9. Sandouk T, Reda D, Hofmann C: The antidiabetic agent pioglitazone content (9,10). In mature adipocytes, troglitazone has also increases expression of glucose transporters in 3T3–F442A cells by been reported to increase GLUT4 mRNA levels (11). These increasing messenger ribonucleic acid transcript stability. Endocrinology in vitro and in vivo studies could not distinguish between 133:352–359, 1993 the increases in glucose transporters associated with the 10. Tafuri SR: Troglitazone enhances differentiation, basal glucose uptake, and Glut1 protein levels in 3T3–L1 adipocytes. Endocrinology 137:4706–4712, enhancement of adipocyte differentiation and those result- 1996 ing from the direct effects of the thiazolidinedione on 11. Tanaka T, Ito H, Doi K, Hosoda K, Shintani M, Yamashita J, Chun TH, Inoue mature adipocytes. In the present study, however, we M, Masatsugu K, Sawada N, Saito T, Inoue G, Nishimura H, Yoshimasa Y, confirmed the increase in GLUT4 protein levels in mature Nakao K: Down regulation of peroxisome proliferator-activated receptor gamma expression by inflammatory cytokines and its reversal by thiazo- adipocytes. The size and morphology of the cells were not lidinediones. Diabetologia 42:702–710, 1999 significantly changed by troglitazone treatment after 24 h 12. Bahr M, Spelleken M, Bock M, Holtey M, Kiehn R, Eckel J: Acute and (data not shown). Our results suggest that troglitazone has chronic effects of troglitazone (CS-045) on isolated rat ventricular cardio- direct effects on mature adipocytes other than adipocyte myocytes. Diabetologia 39:766–774, 1996 differentiation. 13. Yonemitsu S, Nishimura H, Shintani M, Inoue R, Yamamoto Y, Masuzaki H, Ogawa Y, Hosoda K, Inoue G, Hayashi T, Nakao K: Troglitazone induces GLUT4 translocation in L6 myotubes. Diabetes 50:1093–1101, 2001 ACKNOWLEDGMENTS 14. Nishimura H, Zarnowski MJ, Simpson IA: Glucose transporter recycling in This work was supported in part by research grants from rat adipose cells: effects of potassium depletion. J Biol Chem 268:19246– 19253, 1993 the Japanese Ministry of Education, Science and Culture, 15. Choi T, Huang M, Gorman C, Janesch R: A genetic intron increases gene the Japanese Ministry of Health and Welfare, and the expression in transgenic mice. 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