Acute effects of troglitazone and nitric oxide on glucose uptake in L929 fibroblast cells

The thiazolidinedione class of antidiabetic drugs, including troglitazone, has an insulin-sensitizing effect for patients with type 2 diabetes. However, in some tissues, studies have shown that troglitazone also has an acute insulin-independent effect on glucose uptake. To determine the extent of this acute action of troglitazone, the effect of troglitazone on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells was measured. Troglitazone stimulated 2DG uptake in a dose dependent manner with a maximum stimulation of >300% at 5-10 microM. In addition, nitric oxide has been shown to stimulate glucose uptake in peripheral muscle tissue. Therefore, the effect of nitric oxide on 2DG uptake in L929 cells was also investigated using the nitric oxide donor, sodium nitroprusside (SNP). SNP stimulated 2DG uptake by >200% with a maximally effective concentration of 5 mM. The combined effect of maximally effective concentrations of both stimulants (10 microM troglitazone + 5 mM SNP) was not additive suggesting a shared pathway for 2DG uptake. However, the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 50 microM) had no effect on troglitazone stimulated 2DG uptake, indicating that the troglitazone and nitric oxide pathways converge after nitric oxide production. In addition, 12.5 microM dantrolene was shown to have no effect on either troglitazone or SNP stimulated 2DG uptake suggesting that these stimulatory effects are independent of changes in calcium ion concentrations. These data provide important evidence for the acute regulation of glucose transport through GLUT 1 transporters.     

Structurally diverse peroxisome proliferator-activated receptor agonists induce apoptosis in human uro-epithelial cells by a receptor-independent mechanism involving store-operated calcium channels

Objectives:  Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation.
Materials and methods:  Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARγ), ragaglitazar (PPARα/γ), fenofibrate (PPARα) and L165041 (PPARβ/δ).
Results:  NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARγ-mediated apoptosis was unaffected following pre-treatment with PPARγ antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365.
Conclusions:  Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.     

Troglitazone inhibits endothelial cell proliferation through suppression of casein kinase 2 activity

Troglitazone, an agonist of peroxisome proliferator activated receptor gamma (PPARgamma), has been reported to inhibit endothelial cell proliferation by suppressing Akt activation. Recently, it has been also proposed that phosphatase and tensin homolog deleted from chromosome 10 (PTEN) plays an important role in such effect of troglitazone. However, the mechanism of how troglitazone regulates PTEN remains to be elucidated. We therefore investigated the effects of troglitazone on casein kinase 2 (CK2), which is known to negatively regulate PTEN activity. Troglitazone significantly inhibited serum-induced proliferation of HUVEC in a concentration dependent manner. Serum-induced Akt and its downstream signaling pathway activation was attenuated by troglitazone (10 microM) pretreatment. The phosphorylation of PTEN, which was directly related to Akt activation, was decreased with troglitazone pretreatment and was inversely proportional to CK2 activity. DRB, a CK2 inhibitor, also showed effects similar to that of troglitazone on Akt and its downstream signaling molecules. In conclusion, our results suggest that troglitazone inhibits proliferation of HUVECs through suppression of CK2 activity rendering PTEN to remain activated, and this effect of troglitazone in HUVECs seems to be PPARgamma independent.     

Effect of thiazolidinediones on bile acid transport in rat liver

The thiazolidinedione derivatives, troglitazone, rosiglitazone, and pioglitazone, are novel insulin-sensitizing drugs that are useful in the treatment of type 2 diabetes. However, hepatotoxicity associated with troglitazone led to its withdrawal from the market in March 2000. In view of case reports of hepatotoxicity from rosiglitazone and pioglitazone, it is unclear whether thiazolidinediones as a class are associated with hepatotoxicity. Although the mechanism of troglitazone-associated hepatotoxicity has not been elucidated, troglitazone and its major metabolite, troglitazone sulfate, competitively inhibit adenosine triphosphate (ATP)-dependent taurocholate transport in isolated rat canalicular liver plasma membrane vesicles mediated by the canalicular bile salt export pump (Bsep). These results suggest that cholestasis may be a factor in troglitazone-associated hepatotoxicity. To determine whether this effect is 1) limited to canalicular bile acid transport and 2) is specific to troglitazone, the effect of troglitazone, rosiglitazone, and ciglitazone on bile acid transport was examined in rat basolateral (blLPM) and canalicular (cLPM) liver plasma membrane vesicles. In cLPM vesicles, troglitazone, rosiglitazone, and ciglitazone (100 microM) all significantly inhibited ATP-dependent taurocholate transport. In blLPM vesicles, these three thiazolidinediones also significantly inhibited Na(+)-dependent taurocholate transport. Inhibition of bile acid transport was concentration dependent and competitive in both cLPM and blLPM vesicles. In conclusion, these findings are consistent with a class effect by thiazolidinediones on hepatic bile acid transport. If hepatotoxicity is associated with this effect, then hepatotoxicity is not limited to troglitazone. Alternatively, if hepatotoxicity is limited to troglitazone, other mechanisms are responsible for its reported hepatotoxicity.     

Troglitazone acutely activates AMP-activated protein kinase and inhibits insulin secretion from beta cells

Changes in AMP-activated protein kinase (AMPK) activity contribute to the regulation of insulin secretion. Troglitazone has been shown to lower serum insulin levels and protect beta cell function. The aim of the present study was to examine the effects of troglitazone on AMPK activity and insulin secretion in beta cells. Isolated rat islets and MIN6 cells were treated for a short (1 h) or a long time (20 h) with troglitazone. One-hour troglitazone treatment activated AMPK and inhibited both glucose-stimulated insulin secretion (GSIS) and the response of insulin secretion to combined stimuli of glucose and palmitate. Long (20 h) treatment with troglitazone caused a sustained phosphorylation of AMPK and acetyl-CoA carboxylase, and increased GSIS after withdrawal of the drug. This study provided evidence that troglitazone activated AMPK in beta cells. In addition to the insulin-sensitizing effects in peripheral tissues, troglitazone also directly inhibits insulin hypersecretion by the elevated glucose and fatty acids, and thus protects beta cells from glucolipotoxicity.     

Troglitazone suppresses cell growth of myeloid leukemia cell lines by induction of p21WAF1/CIP1 cyclin-dependent kinase inhibitor.

In a human eosinophilic leukemia cell line, EoL-1, cell proliferation was suppressed by 2-day treatment with troglitazone. EoL-1 cells treated with troglitazone were arrested and maintained in the G0/G1 phase in the cell cycle. This suppression correlated with the up-regulation of mRNA for p21WAF1/CIP1 cyclin-dependent kinase (Cdk) inhibitor. The inhibitory effects of troglitazone on cell proliferation and expression of p21 mRNA were observed in a human myelomonocytic cell line, U937, and a human myelomonoblastic cell line, KPB-M15. In addition, in EoL-1 cells, p21 protein was induced by troglitazone treatment and the induction was inhibited by protein synthesis inhibitor, cycloheximide. These data suggest that troglitazone inhibits cell proliferation in myeloid leukemia cell lines at least in part by induction of p21 Cdk inhibitor.     

Connective tissue growth factor and fibronectin secretion in renal tubular epithelial cells induced by TGF-beta1: suppressive effects of troglitazone

Although some studies have suggested that troglitazone could retard the progression of glomerulosclerosis, its effects on renal tubulointerstitial fibrosis have not been completely clarified. The aim of this study was to investigate the effects of troglitazone on the secretion of connective tissue growth factor (CTGF) and fibronectin (FN) in human renal proximal tubular epithelial (HK-2) cells induced by transforming growth factor-beta1 (TGF-beta1). The mRNA of CTGF and FN were measured by semi-quantitative RT-PCR. CTGF and FN protein were detected by Western blot and ELISA, respectively. Our results revealed that troglitazone could inhibit CTGF and FN expression in a dose-dependent manner in human renal proximal tubular epithelial cells induced by TGF-beta1, which may be one of the mechanisms of troglitazone contributing to retard the progression of renal tubulointerstitial fibrosis.     

Differential modulation of COX-2 expression in A549 airway epithelial cells by structurally distinct PPAR(gamma) agonists: evidence for disparate functional effects which are independent of NF-(kappa)B and PPAR(gamma)

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.     

Combined effects of troglitazone and muscle contraction on insulin sensitization in Balb-c mouse muscle

Thiazolidinediones, represented by troglitazone, are insulin-sensitizing agents with proven efficacy for the treatment of type 2 diabetes. Exercise is also recommended for patients with type 2 diabetes because it both stimulates glucose uptake directly and it increases insulin sensitivity following exercise. The purpose of this study was to investigate the effects of troglitazone combined with exercise on 2-deoxyglucose (2DG) uptake in both the epitrochlearis and soleus muscle of Balb-c mice. Acute, 1-h treatment with troglitazone (10 or 20 microM), in the presence or absence of insulin, had no effect on 2DG uptake in either muscle. Chronic treatment with troglitazone by feeding enhanced the insulin sensitivity and responsiveness of 2DG uptake primarily in the epitrochlearis. Direct electrical stimulation of in situ muscle was used to model exercise while the contralateral muscle was used as the unexercised control. This model mimicked exercise in that glycogen was depleted, immediate 2DG uptake was enhanced, and there was a post-exercise increase in insulin sensitivity. Troglitazone feeding had no effect on 2DG uptake in the soleus when measured immediately after electrical stimultion. However, 2DG uptake in the unstimulated epitrochlearis from troglitazone-fed mice was elevated when measured immediately after removal such that no additional effects of the electrical stimulation were measured. We found that the insulin-sensitizing effect of troglitazone was not additive to the insulin-sensitizing effect of exercise, which suggests that troglitazone and exercise share similar pathways. A unique finding in this study was the differential response to troglitazone between the epitrochlearis (fast twitch) and the soleus (slow twitch) muscle types. Possible mechanisms are discussed.     

Regulation of lipoprotein lipase gene expression by insulin and troglitazone in rainbow trout (Oncorhynchus mykiss) adipocyte cells in culture

Adipose tissue plays a central role regulating the balance between deposition and mobilization of lipid reserves. Lipoprotein lipase (LPL) is a key enzyme controlling lipid accumulation in mammals and fish. In the present study, we have examined the expression of LPL in rainbow trout cultured adipocytes and we have investigated the effect of troglitazone, a member of thiazolidinediones (TZDs), and insulin on its expression. LPL gene expression increased from day 1 until day 12 of culture, and the level was maintained up to day 21. The addition of insulin at 10 nM and 1.7 μM increased significantly LPL gene expression in undifferentiated cells (days 7 to 12 maintained in growth medium). Nevertheless, treatment of day 7 cells incubated in growth medium with troglitazone (5 μM) or troglitazone plus insulin (1 μM each), tended to enhance LPL expression. In addition, LPL mRNA levels increased significantly in the presence of 1 μM and 5 μM of troglitazone (days 7 to 12) when the cells were induced to differentiate by addition of differentiation medium. Although troglitazone alone (1 μM) did not stimulate lipid accumulation in the cells neither in growth nor in differentiation medium, the simultaneous presence of troglitazone (1 μM) and insulin (1 μM) increased significantly the content of triglycerides in adipocyte cells maintained in growth medium (days 7 to 12). These results indicate that insulin and troglitazone regulate LPL gene expression during adipocyte differentiation and suggest that both factors may have combined effects in the modulation of adipogenesis.     

Troglitazone has a reducing effect on thromboxane production

Effects of troglitazone, an antidiabetic thiazolidinedione that enhances insulin sensitivity, on thromboxane (TX) production were assessed in human erythroleukemia (HEL) cells and human platelets. Measurement of TX was performed by using the gas chromatography/selected ion monitoring (GC/SIM) method. We found that troglitazone reduced the TX production from HEL cells and human platelets. Furthermore, troglitazone also reduced arachidonic acid (AA)-induced TX production from HEL cell and thrombin-induced TX release from platelets. In addition, we compared the effect of troglitazone with that of alpha-tocopherol and BRL 49653. Other thiazolidinedione compound BRL 49653 had effects similar to troglitazone, but alpha-tocopherol had no effect on TX production. Our findings suggest that the thiazolidinedione group had an antithrombotic effect and was beneficial in preventing vascular complications often observed in diabetes mellitus.     

Troglitazone inhibits expression of the phosphoenolpyruvate carboxykinase gene by an insulin-independent mechanism.

Troglitazone is an oral insulin-sensitizing drug used to treat patients with type 2 diabetes. A major feature of this hyperglycemic state is the presence of increased rates of hepatic gluconeogenesis, which troglitazone is able to ameliorate. In this study, we examined the molecular basis for this property of troglitazone by exploring the effects of this compound on the expression of the two genes encoding the major regulatory enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary cultures of rat hepatocytes. Insulin is able to inhibit expression of both of these genes, which was verified in our model system. Troglitazone significantly reduced mRNA levels of PEPCK and G6Pase in rat hepatocytes isolated from normal and Zucker-diabetic rats, but to a lesser extent than that observed with insulin. Interestingly, troglitazone was unable to reduce cAMP-induced levels of PEPCK mRNA, suggesting that the molecular mechanism whereby troglitazone exerted its effects on gene expression differed from that of insulin. This was further supported by the observation that troglitazone was able to reduce PEPCK mRNA levels in the presence of the insulin signaling pathway inhibitors wortmannin, rapamycin, and PD98059. These results indicate that troglitazone can regulate the expression of specific genes in an insulin-independent manner, and that genes encoding gluconeogenic enzymes are targets for the inhibitory effects of this drug.     

PPAR gamma ligand troglitazone lowers cholesterol synthesis in HepG2 and Caco-2 cells via a reduced concentration of nuclear SREBP-2

Cholesterol synthesis in animal cells is regulated by sterol regulatory element-binding protein (SREBP)-2. The objective of this study was to investigate whether activation of peroxisome proliferator-activatedreceptor (PPAR)-gamma influences the SREBP-2 dependent cholesterol synthesis in liver and intestinal cells. Therefore, HepG2 and Caco-2 cells were incubated with and without 10 or 30 microM of troglitazone, a synthetic PPAR gamma agonist, for 4 hrs. Incubation with 10 or 30 microM of troglitazone caused a significant, dose-dependent reduction of cholesterol synthesis in both HepG2 and Caco-2 cells (P < 0.05). HepG2 and Caco-2 cells incubated with 10 or 30 microM of troglitazone had also lower mRNA concentrations and lower nuclear protein concentrations of SREBP-2 than untreated control cells (P < 0.05). mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase and LDL receptor were also reduced in HepG2 and Caco-2 cells treated with 30 microM of troglitazone compared to control cells (P < 0.05). In conclusion, this study shows that PPAR gamma activation by troglitazone lowers the cholesterol synthesis in HepG2 and Caco-2 cells by reducing the concentration of nuclear SREBP-2 and successive downregulation of its target genes involved in cholesterol synthesis.     

Toxic effect of troglitazone on cultured rat hepatocytes

We examined the cytotoxicity of troglitazone toward cultured rat hepatocytes. The drug concentration- and time-dependently decreased cell viability and increased lactate dehydrogenase leakage from the cells. Troglitazone-induced cell death was characterized by "DNA ladders", condensation of nuclei, and a positive reaction to in situ nick-end labeling. The results indicate that troglitazone can cause apoptotic cell death in cultured rat hepatocytes.     

Ligand activation of peroxisome proliferator-activated receptor gamma induces apoptosis of leukemia cells by down-regulating the c-myc gene expression via blockade of the Tcf-4 activity

The peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor superfamily, is expressed at highest levels in adipose tissue and functions as a central regulator in the process of adipocyte differentiation. In the present study, we showed that human leukemic cell lines, not only myeloid but also lymphoid, express PPAR gamma and its activation by natural ligand (15-deoxy-Delta(12,14) - prostaglandin J(2)) and synthetic ligand (troglitazone) profoundly inhibited their proliferation by induction of apoptosis preferentially in the serum-free culture. We pursued its mechanism using the representative cell lines, and found that induction of apoptosis was accompanied by caspase-3 activation and specifically blocked by its inhibitor. While status of several apoptosis-related molecules remained unchanged, the c-Myc expression was markedly down-regulated within 24 h after troglitazone treatment. The c-myc mRNA levels were dramatically reduced at 1 h and became undetectable at 12 h after troglitazone treatment, which proved to be accompanied by complete blockade of the Tcf-4 activity in the electrophoretic mobility shift assay. We succeeded in establishing HL-60 cell lines growing well in the presence of troglitazone in the long-term serum-free culture. They showed neither induction of apoptosis nor down-regulation of the c-Myc expression via blockade of the Tcf-4 activity after troglitazone treatment. This is the first identification of the linkage between PPAR gamma-mediated apoptosis and down-regulation of the c-myc gene expression.     

Cytotoxicity and apoptosis produced by troglitazone in human hepatoma cells.

Troglitazone is an antidiabetic agent that increases the insulin sensitivity of target tissues in non-insulin-dependent diabetes mellitus. It has been reported that troglitazone causes severe hepatic injury in certain individuals. In the present study, the mechanism for the hepatic injury by troglitazone was investigated with human hepatoma cell lines. HepG2 cells were incubated with troglitazone, its metabolites M-1 (sulfate), M-2 (gulucronide), M-3 (quinone), and other thiazolidinediones (pioglitazone and rosiglitazone). Troglitazone exhibited time- and concentration-dependent cytotoxicity and M-3 also exhibited weak cytotoxicity. Troglitazone induced apoptotic cell death characterized by internucleosomal DNA fragmentation and nuclear condensation. As other thiazolidinediones, pioglitazone and rosiglitazone, did not induce cell death and apoptosis in the present study, the affinity to PPARgamma may not affect the induction of apoptosis by troglitazone. These results suggest that troglitazone induces apoptotic hepatocyte death which it may be one of the factors of liver injury in humans.     

Dietary troglitazone decreases oxidative stress in early stage type II diabetic rats

Oxidative stress is involved in the initiation and development of atherosclerosis in diabetes. We tested the hypothesis that oxidative stress is already increased in early stage type II diabetes, and that troglitazone may prevent the increase. Three groups of 20 week old rats were studied: untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as an animal model of type II diabetes, OLETF rats treated with troglitazone, and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma lipid hydroperoxides (LOOH) concentration, as an indication of lipid peroxidation, and superoxide dismutase (SOD) activity in the thoracic aorta were measured. Plasma LOOH concentration was significantly higher in non-treated OLETF rats compared to LETO rats and treatment with troglitazone completely prevented this increase. SOD activity was significantly decreased in non-treated OLETF rats compared to LETO rats and troglitazone attenuated the diminution of it. These observations demonstrate oxidative stress is already increased in the early stage of type II diabetes and we confirmed troglitazone has the effect of an antioxidant in vivo.     

Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: evidence for involvement of peroxisome proliferator-activated receptor (PPAR) gamma-dependent and PPARgamma-independent signaling pathways

Recently, peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARgamma ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase ( approximately 3.1-fold) was achieved with 20 microm troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 microm treatment for 12 h) significantly increased both the phosphorylation of Ser(1179) of eNOS (eNOS-Ser(1179)) and the dephosphorylation of eNOS-Ser(116) but did not alter eNOS-Thr(497) phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser(1179) with no alteration in eNOS-Ser(116) dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARgamma antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation but with no alteration in eNOS-Ser(116) dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARgamma-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser(1179) phosphorylation and PPARgamma-independent, eNOS-Ser(116) dephosphorylation.     

Troglitazone administration limits infarct size by reduced phosphorylation of canine myocardial connexin43 proteins

Troglitazone, an antidiabetic thiazolidinedione, has been shown to have a scavenging effect on reactive oxygen species, which can modulate expression of connexin43. The study purpose was to evaluate whether troglitazone provides cardioprotection and to assess whether the cardioprotection is associated with an attenuated expression of connexin43 at the border of infarction in a canine model of acute myocardial infarction. Vehicle or troglitazone (1, 5, and 50 mg/kg; n = 14 for each group) was given intravenously 15 min before the coronary artery occlusion. Among the survivors, infarct size was significantly larger in the control than in the supplemented groups. There was a significantly lower infarct size in the high-dose group compared with that in the low-dose group (15 +/- 7% vs. 23 +/- 10% of the risk region in the low-dose group, P = 0.04). Reperfusion caused a significant elevation in superoxide anions as measured by lucigenin-derived chemiluminescence, which was significantly inhibited in animals treated with troglitazone. Connexin43 underwent dephosphorylation in response to ischemia-reperfusion measured by Western blot in control hearts at the border zone; these changes were significantly enhanced by troglitazone administration. Confocal microscopy confirmed the changes of junctional complexes. The magnitude of infarct size positively correlated with the magnitude of phosphorylated connexin43 expression assessed by Western blot analysis (r = 0.73, P < 0.0001). This result demonstrated that the cardioprotective effect of troglitazone as an antioxidant may be associated with reduced phosphorylation of myocardial connexin43 protein.     

Induction of G1 phase arrest and apoptosis in MDA-MB-231 breast cancer cells by troglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligand

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell proliferation and induce apoptosis in cancer cells. Here we wished to determine whether the PPARgamma ligand induces apoptosis and cell cycle arrest of the MDA-MB-231 cell, an estrogen receptor alpha negative breast cancer cell line. The treatment of MDA-MB-231 cell with PPARgamma ligands was shown to induce inhibition of cell growth in a dose-dependent manner as determined by MTT assay. Cell cycle analysis showed a G1 arrest in MDA-MB-231 cells exposed to troglitazone. An apoptotic effect by troglitazone demonstrated that apoptotic cells elevated by 2.5-fold from the control level at 10 microM, to 3.1-fold at 50 microM and to 3.5-fold at 75 microM. Moreover, troglitazone treatment, applied in a dose-dependent manner, caused a marked decrease in pRb, cyclin D1, cyclin D2, cyclin D3, Cdk2, Cdk4 and Cdk6 expression as well as a significant increase in p21 and p27 expression. These results indicate that troglitazone causes growth inhibition, G1 arrest and apoptotic death of MDA-MB-231 cells.