Gene Expression Changes Induced by PPAR Gamma Agonists in Animal and Human Liver

Thiazolidinediones are a class of Peroxisome Proliferator Activated Receptor γ (PPARγ) agonists that reduce insulin resistance in type 2 diabetic patients. Although no detectable hepatic toxicity has been evidenced in animal studies during preclinical trials, these molecules have nevertheless induced hepatic adverse effects in some treated patients. The mechanism(s) of hepatotoxicity remains equivocal. Several studies have been conducted using PCR analysis and microarray technology to identify possible target genes and here we review the data obtained from various in vivo and in vitro experimental models. Although PPARγ is expressed at a much lower level in liver than in adipose tissue, PPARγ agonists exert various PPARγ-dependent effects in liver in addition to PPARγ-independent effects. Differences in effects are dependent on the choice of agonist and experimental conditions in rodent animal studies and in rodent and human liver cell cultures. These effects are much more pronounced in obese and diabetic liver. Moreover, our own recent studies have shown major interindividual variability in the response of primary human hepatocyte populations to troglitazone treatment, supporting the occurrence of hepatotoxicity in only some individuals.

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title: Overlapping Transcriptional Programs Regulated by the Nuclear Receptors Peroxisome Proliferator-Activated Receptor α, Retinoid X Receptor, and Liver X Receptor in Mouse Livers⃞
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title: Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.
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title: Prediction of PPAR-α ligand-mediated physiological changes using gene expression profiles Published, JLR Papers in Press, December 16, 2003. DOI 10.1194/jlr.M300239-JLR200
abstract: Peroxisome proliferator-activated receptor (PPAR)-α controls the transcription of a variety of genes involved in lipid metabolism and is the target receptor for the hypolipidemic drug class of fibrates. In the present study, the molecular and physiological effects of seven different PPAR-activating drugs have been examined in a rodent model of dyslipidemia. The drugs examined were selected to display varying potencies and efficacies toward PPAR-α. To help elucidate the link between the gene regulation elicited by PPAR-α ligands and the concomitant physiological changes, we have used cDNA microarray analysis to identify smaller gene sets that are predictive of the function of these ligands. A number of genes showed strong correlations to the relative PPAR-α efficacy of the drugs. Furthermore, using multivariate analysis, a strong relationship between the drug-induced triglyceride lowering and the transcriptional profiles of the different drugs could be found.

title: Up-Regulation of Peroxisome Proliferator-Activated Receptors (PPAR-α) and PPAR-γ Messenger Ribonucleic Acid Expression in the Liver in Murine Obesity: Troglitazone Induces Expression of PPAR-γ-Responsive Adipose Tissue-Specific Genes in the Liver of Obese Diabetic Mice* * This work was supported by 
abstract: Peroxisome proliferator-activated receptors (PPARs) are transcription factors that play an important role in the regulation of genes involved in lipid utilization and storage, lipoprotein metabolism, adipocyte differentiation, and insulin action. The three isoforms of the PPAR family, i.e. alpha, delta, and gamma, have distinct tissue distribution patterns. PPAR-alpha is predominantly present in the liver, and PPAR-gamma in adipose tissue, whereas PPAR-delta is ubiquitously expressed. A recent study reported increased PPAR-gamma messenger RNA (mRNA) expression in the liver in ob/ob mice; however, it is not known whether increased PPAR-gamma expression in the liver has any functional consequences. The expression of PPAR-alpha and -delta in the liver in obesity has not been determined. We have now examined the mRNA levels of PPAR-alpha, -delta, and -gamma in three murine models of obesity, namely, ob/ob (leptin-deficient), db/db (leptin-receptor deficient), and serotonin 5-HT2c receptor (5-HT2cR) mutant mice. 5-HT2cR mutant mice develop a late-onset obesity that is associated with higher plasma leptin levels. Our results show that PPAR-alpha mRNA levels in the liver are increased by 2- to 3-fold in all three obese models, whereas hepatic PPAR-gamma mRNA levels are increased by 7- to 9-fold in ob/ob and db/db mice and by 2-fold in obese 5-HT2cR mutant mice. PPAR-delta mRNA expression is not altered in ob/ob or db/db mice. To determine whether increased PPAR-gamma expression in the liver has any functional consequences, we examined the effect of troglitazone treatment on the hepatic mRNA levels of several PPAR-gamma-responsive adipose tissue-specific genes that have either no detectable or very low basal expression in the liver. The treatment of lean control mice with troglitazone significantly increased the expression of adipocyte fatty acid-binding protein (aP2) and fatty acid translocase (FAT/CD36) in the liver. This troglitazone-induced increase in the expression of aP2 and FAT/CD36 was markedly enhanced in the liver in ob/ob mice. Troglitazone also induced a pronounced increase in the expression of uncoupling protein-2 in the liver in ob/ob mice. In contrast to the liver, troglitazone did not increase the expression of aP2, FAT/CD36, and uncoupling protein-2 in adipose tissue in lean or ob/ob mice. Taken together, our results suggest that the effects of PPAR-gamma activators on lipid metabolism and energy homeostasis in obesity and type 2 diabetes may be partly mediated through their effects on PPAR-gamma in the liver.

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abstract: Peroxisome proliferators are a diverse group of compounds that cause hepatic hypertrophy and hyperplasia, increase peroxisome number, and on chronic high-dose administration, lead to rodent liver tumorigenesis. Various lines of evidence have led to the conclusion that these agents induce their pleiotropic effects exclusively via agonism of peroxisome proliferator-activated receptor (PPAR)alpha, a member of the steroid receptor superfamily involved in the regulation of fatty acid metabolism. Recently, agonists of two other members of this receptor family have been identified. PPARgamma is predominantly expressed in adipocytes where it mediates differentiation; PPARdelta is a widely expressed orphan receptor with yet unresolved physiologic functions. In the course of characterizing newer PPAR ligands, we noted that highly selective PPARgamma agonists or dual PPARgamma/PPARdelta agonists, lacking apparent murine PPARalpha agonist activity, cause peroxisome proliferation in CD-1 mice. We therefore made use of PPARalpha knockout mice to investigate whether these effects resulted from agonism of PPARalpha by these agents at very high dose levels or whether PPARgamma (or PPARdelta) agonism alone can result in peroxisome proliferation. We report here that several parameters linked to the hepatic peroxisome proliferation response in mice that were seen with these agents resulted from PPARalpha-independent effects.

title: Differential activation of adipogenesis by multiple PPAR isoforms.
abstract: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear hormone receptor expressed predominantly in adipose tissue, where it plays a central role in the control of adipocyte gene expression and differentiation. Because there are two additional PPAR isoforms, PPARalpha and PPARdelta, and these are also expressed at some level in certain adipose depots, we have compared directly the adipogenic potential of all three receptors. Ectopically expressed PPARgamma powerfully induces adipogenesis at a morphological and molecular level in response to a number of PPARgamma activators. PPARalpha is less adipogenic but is able to induce significant differentiation in response to strong PPARalpha activators. Expression and activation of PPARdelta did not stimulate adipogenesis. Of the three PPARs, only PPARgamma can cooperate with C/EBPalpha in the promotion of adipogenesis. To begin to investigate the functional basis for the differential adipogenic activity of the PPAR isoforms, we have examined their ability to bind to several PPAR DNA response sequences. Compared with PPARalpha and PPARdelta, PPARgamma shows preferential binding to two well-characterized regulatory sequences derived from a fat-specific gene, ARE6 and ARE7. These data strongly suggest that PPARgamma is the predominant receptor regulating adipogenesis; however, they also suggest that PPARalpha may play a role in differentiation of certain adipose depots in response to a different set of physiologic activators or in certain disease states.

title: Troglitazone enhances the hepatotoxicity of acetaminophen by inducing CYP3A in rats.
abstract: Troglitazone (TRZ) is the first of a new group of oral antidiabetic drugs, the thiazolidinediones, and is proven to lower plasma glucose levels in patients with type 2 diabetes mellitus. However, the concern has been raised because of several reports, in which severe hepatic dysfunction leading to hepatic failure was demonstrated in a few patients receiving the drug. We studied the effects of TRZ on the hepatotoxicity of carbon tetrachloride (CCl(4)) and acetaminophen (APAP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 3A (CYP3A) and 2E1 (CYP2E1). Male standard (Wistar/ST) and type 2 diabetic model (GK/Jal) rats were kept on a powdered chow diet containing 0, 100, 500 mg/kg/rat of TRZ. Three weeks later, the rats were either sacrificed for an in vitro metabolism study or challenged with 0.50 g/kg CCl(4) p.o. or 0.75 g/kg APAP i.p.TRZ at 100 and 500 mg/kg/rat increased the CYP3A level as well as the testosterone 6beta-hydroxylation activities in liver microsomes, but did not affect CYP2E1. TRZ also enhanced APAP hepatotoxicity, as evidenced by significantly increased levels of alanine aminotransferase, aspartate aminotransferase and alpha-glutathione S-transferase in the plasma of rats, and by significantly low hepatic glutathione concentration. Our study demonstrated that high doses of TRZ can enhance hepatotoxicity of APAP in Wistar/ST and GK/Jal by inducing hepatic CYP3A.

title: Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: in vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat.
abstract: Troglitazone is a thiazolidinedione insulin sensitizer drug for the treatment of type 2 non-insulin-dependent diabetes mellitus (NIDDM). Based on an increasing number of reports on troglitazone-associated liver toxicity, the cholestatic potential of troglitazone has been investigated. Rapid and dose-dependent increases in the plasma bile acid concentrations were observed in rats after a single intravenous administration of troglitazone. A radiolabeled taurocholic acid tracer accumulated in liver tissue, indicating an interference with the hepatobiliary export of bile acids. In isolated canalicular rat liver plasma membrane preparations, troglitazone competitively inhibited the ATP-dependent taurocholate transport (apparent K(i) value, 1.3 microM), mediated by the canalicular bile salt export pump (Bsep). Troglitazone sulfate, the main troglitazone metabolite eliminated into bile, also showed competitive Bsep inhibition with an apparent K(i) value of 0.23 microM. A comparable inhibition was observed for both compounds in canalicular plasma membrane vesicles prepared from Mrp2-deficient (TR(-)) rats, suggesting a direct (cis-) inhibition of Bsep by troglitazone and troglitazone sulfate. A high accumulation potential was observed for troglitazone sulfate in rat liver tissue, indicating that the hepatobiliary export of this conjugated metabolite might represent a rate-limiting step in the overall elimination process of troglitazone. This accumulation in combination with the high Bsep inhibition potential suggested that mainly troglitazone sulfate was responsible for the interaction with the hepatobiliary export of bile acids at the level of the canalicular Bsep in rats. Such an interaction might lead to a troglitazone-induced intrahepatic cholestasis in humans as well, contributing to the formation of a troglitazone-induced liver toxicity.

title: Mechanisms of troglitazone hepatotoxicity.
abstract: In January 1997, the drug Rezulin (TGZ)1 was approved for use in the treatment of type II diabetes by the Food and Drug Administration. Between one and two million patients were treated with TGZ from 1997 through the end of February 2000, when TGZ was withdrawn from the market (1). This withdrawal was based on numerous reports of liver failure associated with TGZ use. For example, Kohlroser et al. reviewed 46 cases of hepatotoxicity among Rezulin users and concluded that “there can be no doubt that, in some patients, TGZ causes severe hepatotoxicity” (2). The exact nature of the hepatotoxicity remained controversial, however. Initial reports described TGZ hepatotoxicity as being idiosyncratic, that is, a rare event that occurs without explanation and is not dose-dependent. Usually such hepatotoxins work by an indirect, often immune, allergic type mechanism. However, in clinical trials, more than 1.9% of patients had elevated serum transaminases and other signs of liver toxicity. Thus, the moderate hepatotoxic effects of TGZ were not that rare. Furthermore, few, if any, of the patients with severe liver damage showed signs of fever or other indicators of a cytotoxic lymphocyte-based immune mechanism. Moreover, TGZ often produced a zonal necrosis of the liver, whereas idiosyncratic hepatotoxins usually produce a diffuse, focal necrosis. Zonal necrosis of the centrilobular region is usually consistent with metabolism of a hepatotoxin to reactive intermediates that produce direct toxic effects. Considerable evidence has also emerged that TGZ can be directly toxic to liver cells leading to suggestions that TGZ is “an idiosyncratic direct-acting hepatotoxin” (3). Here, I discuss the possible

title: Troglitazone-induced hepatic mitochondrial proteome expression dynamics in heterozygous Sod2(+/-) mice: two-stage oxidative injury.
abstract: The determinants of susceptibility to troglitazone-induced idiosyncratic liver injury have not yet been determined; however, troglitazone has been shown to target mitochondria and induce mitochondria-mediated hepatocellular injury in vitro. The aim of this study was to use a systems approach to analyze the dynamics of mitochondrial changes at the proteome level and more clearly define the mechanisms and time course of troglitazone hepatotoxicity by using a previously characterized mouse model that is highly sensitized to troglitazone hepatotoxicity. Mice heterozygous in mitochondrial superoxide dismutase-2 (Sod2(+/-)) were injected intraperitoneally with troglitazone (30 mg/kg/day) or vehicle daily for 2 or 4 weeks. Hepatic mitochondria were isolated, purified, and subjected to two-dimensional difference gel electrophoresis (2D-DIGE). We found that among the ~1500 resolved hepatic mitochondrial proteins, 70 exhibited significantly altered abundance after troglitazone treatment. MALDI-TOF/TOF MS/MS analysis revealed that early changes (2 weeks) included increased levels of heat shock protein family members (mortalin, HSP7C), Lon protease, and catalase, indicating induction of a mitochondrial stress response. In contrast, after 4 weeks, a number of critical proteins including ATP synthase beta-subunit, aconitase-2, and catalase exhibited decreased abundance, and total protein carbonyls were significantly increased, suggesting uncompensated oxidative damage. Aconitase-2 (ACO2) was decreased at both time points, making this protein a potential sensitive and early biomarker for mitochondrial oxidant stress. These results show that, in this murine model of underlying clinically silent mitochondrial stress, superimposed troglitazone induces a two-stage response: an initial adaptive response, followed by a toxic response involving oxidant injury to mitochondrial proteins.

title: Down-regulation by troglitazone of hepatic tumor necrosis factor-alpha and interleukin-6 mRNA expression in a murine model of non-insulin-dependent diabetes.
abstract: Troglitazone, a novel thiazolidinedione drug used to treat non-insulin-dependent diabetes mellitus, is a selective ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). Recent results indicate that PPARgamma activation by thiazolidinediones regulates adipose tissue- and monocyte/peritoneal macrophage-derived cytokine expression in vitro. We evaluated whether troglitazone may also negatively regulate cytokine expression in the liver, which harbors the majority of the body's resident macrophages but which only weakly expresses PPARgamma. Lean C57BL6 mice and genetically obese KKA(y) mice were chronically treated with troglitazone (100 mg/kg/day for 2 weeks). At the end of treatment, hepatic expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA was quantitatively determined by kinetic polymerase chain reaction both under basal conditions and after stimulation with lipopolysaccharide (LPS). Both untreated lean and obese mice exhibited low levels of baseline TNF-alpha and IL-6 mRNA expression and responded with a dramatic increase in hepatic cytokine transcripts and TNF-alpha protein expression following a challenge with LPS. Similar to the effects on white adipose tissue, troglitazone not only down-regulated the baseline levels of hepatic TNF-alpha and IL-6, but also greatly attenuated the inducing effects of LPS. The extent of this inhibitory effect of troglitazone was higher in obese KKA(y) mice than in lean mice and was also reflected by markedly down-regulated hepatic TNF-alpha protein expression. These data demonstrate that chronic administration of troglitazone is associated with a greatly attenuated responsiveness towards inducers of hepatic TNF-alpha and IL-6 production. The possible biological consequences of these effects, however, have not yet been assessed.

title: The peroxisome proliferator-activated receptor-γ is a negative regulator of macrophage activation
abstract: The peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors that is predominantly expressed in adipose tissue, adrenal gland and spleen. PPAR-γ has been demonstrated to regulate adipocyte differentiation and glucose homeostasis in response to several structurally distinct compounds, including thiazolidinediones and fibrates. Naturally occurring compounds such as fatty acids and the prostaglandin D2 metabolite 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) bind to PPAR-γ and stimulate transcription of target genes. Prostaglandin D2metabolites have not yet been identified in adipose tissue, butaremajor products of arachidonic-acid metabolism in macrophages, raising the possibility that they might serve as endogenous PPAR-γ ligands in this cell type. Here we show that PPAR-γ is markedly upregulated in activated macrophages and inhibits the expression of the inducible nitric oxide synthase, gelatinase B and scavenger receptor A genes in response to 15d-PGJ2 and synthetic PPAR-γ ligands. PPAR-γ inhibits gene expression in part by antagonizing the activities of the transcription factors AP-1, STAT and NF-κB. These observations suggest that PPAR-γ and locally produced prostaglandin D2 metabolites are involved in the regulation of inflammatory responses, and raise the possibility that synthetic PPAR-γ ligands may be of therapeutic value in human diseases such as atherosclerosis and rheumatoid arthritis in which activated macrophages exert pathogenic effects.

title: Liver Peroxisome Proliferator-activated Receptor γ Contributes to Hepatic Steatosis, Triglyceride Clearance, and Regulation of Body Fat Mass*
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that mediates the antidiabetic effects of thiazolidinediones. PPARγ is present in adipose tissue and becomes elevated in fatty livers, but the roles of specific tissues in thiazolidinedione actions are unclear. We studied the function of liver PPARγ in both lipoatrophic A-ZIP/F-1 (AZIP) and wild type mice. In AZIP mice, ablation of liver PPARγ reduced the hepatic steatosis but worsened the hyperlipidemia, triglyceride clearance, and muscle insulin resistance. Inactivation of AZIP liver PPARγ also abolished the hypoglycemic and hypolipidemic effects of rosiglitazone, demonstrating that, in the absence of adipose tissue, the liver is a primary and major site of thiazolidinedione action. In contrast, rosiglitazone remained effective in non-lipoatrophic mice lacking liver PPARγ, suggesting that adipose tissue is the major site of thiazolidinedione action in typical mice with adipose tissue. Interestingly, mice without liver PPARγ, but with adipose tissue, developed relative fat intolerance, increased adiposity, hyperlipidemia, and insulin resistance. Thus, liver PPARγ regulates triglyceride homeostasis, contributing to hepatic steatosis, but protecting other tissues from triglyceride accumulation and insulin resistance.

title: Diabetic KKAy mice exhibit increased hepatic PPARgamma1 gene expression and develop hepatic steatosis upon chronic treatment with antidiabetic thiazolidinediones.
abstract: Peroxisome proliferator-activated receptor-gamma, which is involved in the regulation of lipid homeostasis, is upregulated in the liver of obese and diabetic mice, but the biological consequences of this induction are largely unknown. This study was aimed at further characterizing this upregulation and exploring the downstream biological effects of specific activators on hepatic lipid metabolism.



Hepatic expression of peroxisome proliferator-activated receptor-gamma1 and gamma2 mRNA and protein was analyzed by real-time polymerase chain reaction and Western immunoblotting in KKAy mice and ob/ob mice. KKAy mice were treated with thiazolidinediones, and hepatic triglyceride content and lipid distribution were analyzed biochemically and by histopathology.



KKAy mice exhibited a marked increase in hepatic peroxisome proliferator-activated receptor-gamma1 mRNA and protein levels, whereas the gamma2 isoform was upregulated in ob/ob mice. Treatment of KKAy mice with troglitazone or rosiglitazone resulted in severe microvesicular periacinar steatosis, whereas lean control mice did not develop any pathological liver changes. Hepatic triglyceride levels, however, were not altered by the treatment.



In mice with obesity-associated upregulated hepatic peroxisome proliferator-activated receptor-gamma expression, thiazolidinediones may produce hepatic steatosis. Under pathophysiological conditions, such as non-insulin-dependent diabetes, the liver may thus become sensitized towards peroxisome proliferator-activated receptor-gamma-activating drugs.

title: Troglitazone action is independent of adipose tissue.
abstract: We have investigated the antidiabetic action of troglitazone in aP2/DTA mice, whose white and brown fat was virtually eliminated by fat-specific expression of diphtheria toxin A chain. aP2/DTA mice had markedly suppressed serum leptin levels and were hyperphagic, but did not gain excess weight. aP2/DTA mice fed a control diet were hyperlipidemic, hyperglycemic, and had hyperinsulinemia indicative of insulin-resistant diabetes. Treatment with troglitazone alleviated the hyperglycemia, normalized the tolerance to intraperitoneally injected glucose, and significantly decreased elevated insulin levels. Troglitazone also markedly decreased the serum levels of cholesterol, triglycerides, and free fatty acids both in wild-type and aP2/DTA mice. The decrease in serum triglycerides in aP2/DTA mice was due to a marked reduction in VLDL- and LDL-associated triglyceride. In skeletal muscle, triglyceride levels were decreased in aP2/DTA mice compared with controls, but glycogen levels were increased. Troglitazone treatment decreased skeletal muscle, but not hepatic triglyceride and increased hepatic and muscle glycogen content in wild-type mice. Troglitazone decreased muscle glycogen content in aP2/DTA mice without affecting muscle triglyceride levels. The levels of peroxisomal proliferator-activated receptor gamma mRNA in liver increased slightly in aP2/DTA mice and were not changed by troglitazone treatment. The results demonstrate that insulin resistance and diabetes can occur in animals without significant adipose deposits. Furthermore, troglitazone can alter glucose and lipid metabolism independent of its effects on adipose tissue.

title: Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes.
abstract: To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.

title: Rosiglitazone (BRL49653), a PPARgamma-selective agonist, causes peroxisome proliferator-like liver effects in obese mice.
abstract: The PPAR (peroxisome proliferator activated receptor) transcription factors are ligand-activated nuclear receptors that regulate genes involved in lipid metabolism and homeostasis. PPARalpha is preferentially expressed in liver and PPARgamma preferentially in adipose tissue. Activation of PPARalpha leads to peroxisome proliferation and increased beta-oxidation of fatty acids in rodents. PPARgamma-activation leads to adipocyte differentiation and improved insulin signaling of mature adipocytes. Both PPAR receptors are believed to be functional targets for treatment of hyperlipidemia in man. We have treated obese diabetic mice (ob/ob), which have highly elevated levels of plasma triglycerides, glucose and insulin, for 1 week with WY14,643 (180 micromol/kg/day), a selective PPARalpha agonist, or rosiglitazone (BRL49653; 2.5 micromol/kg/day), a selective PPARgamma agonist. The doses used produce a similar therapeutic effect in both treatment groups (lowering of triglycerides and glucose). High resolution two-dimensional gel electrophoresis of livers showed that WY14,643 and rosiglitazone both produced changes in expression pattern of many proteins involved in peroxisomal fatty acid beta-oxidation. However, similar experiments performed in lean mice showed significant up-regulation of these proteins only with WY14,643 treatment. Furthermore, the proteins up-regulated by the drugs in obese mice had a higher basal expression in obese controls compared to the lean littermates. Liver PPARgamma mRNA levels were determined and we observed that PPARgamma2 mRNA levels were elevated in obese mice compared to lean littermates. As PPARalpha and PPARgamma recognize similar DNA response elements, it is likely that the effects of rosiglitazone on PPARalpha responsive genes in livers of the ob/ob mice are mediated by PPARgamma2.

title: Adipocyte-specific Gene Expression and Adipogenic Steatosis in the Mouse Liver Due to Peroxisome Proliferator-activated Receptor γ1 (PPARγ1) Overexpression*
abstract: Peroxisome proliferator activated-receptor (PPAR) isoforms, α and γ, function as important coregulators of energy (lipid) homeostasis. PPARα regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPARγ serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPARγ isoforms, PPARγ1 and PPARγ2 generated by alternative splicing, PPARγ1 isoform is expressed in liver and other tissues, whereas PPARγ2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPARγ1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPARγ1 in mouse liver. Adenovirus-PPARγ1 injected into the tail vein induced hepatic steatosis in PPARα−/− mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPARα−/− livers with PPARγ1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, Δ9desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosisper se, induced either by feeding a diet deficient in choline or developing in fasted PPARα−/− mice, failed to induce the expression of these PPARγ-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPARγ in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPARγ activity can lead to the development of a novel type of adipogenic hepatic steatosis.

title: Identification of promethin and PGLP as two novel up-regulated genes in PPARgamma1-induced adipogenic mouse liver.
abstract: Peroxisome proliferator-activated receptor (PPAR) isoforms, alpha, gamma and beta/delta, function as important lipid sensors as well as key regulators of energy homeostasis. PPARalpha plays a dynamic role in energy combustion by transcriptionally upregulating fatty acid oxidation systems primarily in liver, whereas PPARgamma functions as a regulator of adipogenesis and lipid storage. Overexpression of PPARgamma, using adenoviral expression approach, in PPARalpha deficient mouse liver results in hepatic steatosis with concurrent expression of adipocyte specific genes. In this study, to gain a global molecular understanding of PPARgamma1-induced gene expression in liver, we have analyzed gene expression profiles using the Affymetrix GeneChip mouse expression array set 430, that enables a comprehensive gene expression profiling with >39,000 transcripts. Microarray data analysis provided us with over 278 genes up-regulated fourfold or higher, and 121 genes down-regulated fourfold or higher in liver with PPARgamma-induced hepatic adiposis. We have found 101 uncharacterized genes out of 278 up-regulated and 29 uncharacterized among the down-regulated gene categories, respectively. Of 177 functionally characterized candidate genes in the up-regulated category many appear to be involved in adipogenesis, lipid metabolism and signal transduction. To focus attention on the uncharacterized genes in the up-regulated category, we cloned the full-length cDNAs of two novel candidates, which we designated as promethin and PGLP. Promethin, a 15-kDa cytosolic protein, is not normally expressed in liver but induced robustly in liver with hepatic adiposis caused by PPARgamma overexpression. PGLP, which encodes a 38 kDa cytoplasmic membranous protein, is a low abundant transcript in normal liver, but induced dramatically following PPARgamma overexpression. The expression of these two genes was not increased in fatty livers induced by fasting or choline deficiency. The identification of these and other novel PPARgamma-target genes should provide a basis for understanding the molecular mechanisms underlying energy storage and lipid homeostasis.

title: Regulation of PPAR gamma gene expression by nutrition and obesity in rodents.
abstract: The orphan nuclear receptor, peroxisome proliferator-activated receptor (PPAR) gamma, is implicated in mediating expression of fat-specific genes and in activating the program of adipocyte differentiation. The potential for regulation of PPAR gamma gene expression in vivo is unknown. We cloned a partial mouse PPAR gamma cDNA and developed an RNase protection assay that permits simultaneous quantitation of mRNAs for both gamma l and gamma 2 isoforms encoded by the PPAR gamma gene. Probes for detection of adipocyte P2, the obese gene product, leptin, and 18S mRNAs were also employed. Both gamma l and gamma 2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma 1 expression was also detected at lower levels in liver, spleen, and heart; whereas, gamma l and gamma 2 mRNA were expressed at low levels in skeletal muscle. Adipose tissue levels of gamma l and gamma 2 were not altered in two murine models of obesity (gold thioglucose and ob/ob), but were modestly increased in mice with toxigene-induced brown fat ablation uncoupling protein diphtheria toxin A mice. Fasting (12-48 h) was associated with an 80% fall in PPAR gamma 2 and a 50% fall in PPAR gamma mRNA levels in adipose tissue. Western blot analysis demonstrated a marked effect of fasting to reduce PPAR gamma protein levels in adipose tissue. Similar effects of fasting on PPAR gamma mRNAs were noted in all three models of obesity. Insulin-deficient (streptozotocin) diabetes suppressed adipose tissue gamma l and gamma 2 expression by 75% in normal mice with partial restoration during insulin treatment. Levels of adipose tissue PPAR gamma 2 mRNA were increased by 50% in normal mice exposed to a high fat diet. In obese uncoupling protein diphtheria toxin A mice, high fat feeding resulted in de novo induction of PPAR gamma 2 expression in liver. We conclude (a) PPAR gamma 2 mRNA expression is most abundant in adipocytes in normal mice, but lower level expression is seen in skeletal muscle; (b) expression of adipose tissue gamma1 or gamma2 mRNAs is increased in only one of the three models of obesity; (c) PPAR gamma 1 and gamma 2 expression is downregulated by fasting and insulin-deficient diabetes; and (d) exposure of mice to a high fat diet increases adipose tissue expression of PPAR gamma (in normal mice) and induces PPAR gamma 2 mRNA expression in liver (in obese mice). These findings demonstrate in vivo modulation of PPAR gamma mRNA levels over a fourfold range and provide an additional level of regulation for the control of adipocyte development and function.

title: Comprehensive Messenger Ribonucleic Acid Profiling Reveals That Peroxisome Proliferator-Activated Receptor γ Activation Has Coordinate Effects on Gene Expression in Multiple Insulin-Sensitive Tissues.
abstract: Peroxisome proliferator-activated receptor γ (PPARγ) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPARγ ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPARγ in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPARγ activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle...

title: Differential effects of rexinoids and thiazolidinediones on metabolic gene expression in diabetic rodents.
abstract: Both retinoid X receptor (RXR)-selective agonists (rexinoids) and thiazolidinediones (TZDs), PPAR (peroxisome proliferator-activated receptor)-gamma-specific ligands, produce insulin sensitization in diabetic rodents. In vitro studies have demonstrated that TZDs mediate their effects via the RXR/PPAR-gamma complex. To determine whether rexinoids lower hyperglycemia by activating the RXR/PPAR-gamma heterodimer in vivo, we compared the effects of a rexinoid (LG100268) and a TZD (rosiglitazone) on gene expression in white adipose tissue, skeletal muscle, and liver of Zucker diabetic fatty rats (ZDFs). In adipose tissue, rosiglitazone decreased tumor necrosis factor-alpha (TNF-alpha) mRNA and induced glucose transporter 4 (GLUT4), muscle carnitine palmitoyl-transferase (MCPT), stearoyl CoA desaturase (SCD1), and fatty acid translocase (CD36). In contrast, LG100268 increased TNF-alpha and had no effect or suppressed the expression of GLUT4, MCPT, SCD1, and CD36. In liver, the rexinoid increased MCPT, SCD1, and CD36 mRNAs, whereas rosiglitazone induced only a small increase in CD36. In skeletal muscle, rosiglitazone and LG100268 have similar effects; both increased SCD1 and CD36 mRNAs. The differences in the pattern of genes induced by the rexinoids and the TZDs in diabetic animals found in these studies suggests that these compounds may have independent and tissue-specific effects on metabolic control in vivo.

title: Alteration in expression profiles of a series of diabetes-related genes in db/db mice following treatment with thiazolidinediones.
abstract: We studied the effect of pioglitazone on the transcription of 42 genes associated with diabetes to examine the relationship between the antidiabetic action of thiazolidinediones (TZDs) and their ability to modulate transcription through their peroxisome proliferater-activated receptor (PPAR)-agonistic activity. Diabetic (db/db) mice were orally administered with pioglitazone for two weeks. Total RNA was prepared from liver, muscle and adipocytes and the quantity of mRNA was determined by comparative RT-PCR. The expression of diabetes-related genes was compared between lean and untreated db/db mice and between untreated and drug-treated db/db mice. The onset of diabetes was associated with a considerable alteration in the expression of a large number of diabetes-related genes. Treatment of db/db mice with pioglitazone modulated the expression of genes involved in the metabolism of glucose, lipids and lipoproteins. This included genes for phosphoenolpyruvate carboxykinase, beta-oxidation enzymes, lipoprotein lipase, apolipoprotein AI and uncoupling proteins. Most of the genes responsible for insulin signaling were unaffected. Administration of pioglitazone was also shown to induce PPARgamma expression in liver and muscle. It is therefore possible to hypothesize that TZDs may ameliorate diabetes through a mechanism of action involving a direct decrease in plasma glucose and triglyceride levels and improvements in free fatty acid-induced insulin resistance.

title: Insulin sensitization in diabetic rat liver by an antihyperglycemic agent.
abstract: This study aimed to demonstrate directly that the thiazolidinedione pioglitazone acts as an insulin sensitizer. We tested the hypothesis that pioglitazone treatment of diabetic rats alters liver function such that responsiveness of selected genes to subsequent insulin regulation is enhanced. Although flux through gluconeogenic/glycolytic pathways involves regulation of many enzymes, we presently report the effects of insulin on expression of two key enzymes in these metabolic pathways, ie, phosphoenolpyruvate carboxykinase (PEPCK) and glucokinase (GK). Rats were either studied as nondiabetic controls or injected with streptozotocin as a model for insulin-deficient diabetes. Diabetic animals were treated without or with pioglitazone and subsequently examined for acute responses to insulin. Pioglitazone treatment of diabetic animals significantly enhanced the effects of insulin to reverse elevated blood glucose. Although the mean level of liver mRNA transcripts encoding PEPCK was increased to nearly 300% in diabetic animals as compared with nondiabetic controls (100%), it was significantly lower in pioglitazone-treated diabetic rats (119% of control) than in diabetic rats without pioglitazone (223% of control) after insulin treatment. By contrast, mRNA transcripts encoding GK were not detectable in diabetic animals, but were increased markedly by insulin treatment in all animal groups. Insulin-enhanced expression of GK was significantly greater in liver from animals that were treated earlier with pioglitazone (291% of control) than in liver from those that were untreated (214% of control). An amplified acute response of liver to insulin thus established pioglitazone as an insulin sensitizer. Our findings further showed that such sensitization can be developed even in the insulin-deficient state.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Tissue Distribution and Quantification of the Expression of mRNAs of Peroxisome Proliferator–Activated Receptors and Liver X Receptor-α in Humans: No Alteration in Adipose Tissue of Obese and NIDDM Patients
abstract: Members of the peroxisome proliferator–activated receptor (PPAR) family might be involved in pathologies with altered lipid metabolism. They participate in the control of the expression of genes involved in lipid metabolism and adipocyte differentiation. In addition, thiazolidinediones improve insulin resistance in vivo by activating PPARγ. However, little is known regarding their tissue distribution and relative expression in humans. Using a quantitative and sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) assay, we determined the distribution and relative mRNA expression of the four PPARs (α, β, γ1, and γ2) and liver X receptor-α (LXRα) in the main tissues implicated in lipid metabolism. PPARα and LXRα were mainly expressed in liver, while PPARγ1 predominated in adipose tissue and large intestine. We found that PPARγ2 mRNA was a minor isoform, even in adipose tissue, thus causing question of its role in humans. PPARβ mRNA was present in all the tissues tested at low levels. In addition, PPARγ mRNA was barely detectable in skeletal muscle, suggesting that improvement of insulin resistance with thiazolidinediones may not result from a direct effect of these agents on PPARγ in muscle. Obesity and NIDDM were not associated with change in PPARs and LXRα expression in adipose tissue. The mRNA levels of PPARγ1, the predominant form in adipocytes, did not correlate with BMI, leptin mRNA levels, or fasting insulinemia in 29 subjects with various degrees of obesity. These results indicated that obesity is not associated with alteration in PPAR gene expression in abdominal subcutaneous adipose tissue in humans.

title: Beneficial effects of PPAR-γ ligands in ischemia–reperfusion injury, inflammation and shock
abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. PPAR-γ regulates gene expression by forming a heterodimer with the retinoid X receptor (RXR) before binding to sequence-specific PPAR response elements (PPREs) in the promoter region of target genes, thereby regulating several metabolic pathways, including lipid biosynthesis and glucose metabolism. Thiazolidinediones (TZDs, i.e. rosiglitazone, pioglitazone), which are synthetic PPAR-γ agonists, act as insulin sensitizers and are used in the treatment of type 2 diabetes. In the last few years, it has, however, become evident that the therapeutic effects of PPAR-γ ligands reach far beyond their use as insulin sensitizers. Recently, PPAR-γ has been implicated as a regulator of cellular inflammatory and ischemic responses. PPAR-γ agonists may exert their anti-inflammatory effects by negatively regulating the expression of pro-inflammatory genes induced during macrophage differentiation and activation, by either PPAR-γ-dependent or -independent mechanisms. Several lines of evidence suggest that TZDs protect the heart and other organs against the tissue injury caused by ischemia/reperfusion (I/R) injury and shock. This review discusses the anti-inflammatory signalling pathways activated by PPAR-γ, as well as the potential therapeutic effects of PPAR-γ agonists in animal models of ischemia/reperfusion, inflammation and shock.

title: Effects of chronic rosiglitazone therapy on gene expression in human adipose tissue in vivo in patients with type 2 diabetes.
abstract: The aim of this study was to compare effects of therapeutic doses of rosiglitazone and metformin on expression of 50 genes in human adipose tissue in vivo.



Twenty patients with diet-treated type 2 diabetes (13 women, seven men) were randomized to receive either rosiglitazone (n = 9; 8 mg/d) or metformin (n = 11; 2 g/d) for 16 wk. Subcutaneous adipose tissue biopsies were performed before and after treatment. Expression of 50 genes, previously shown to be altered by thiazolidinediones in experimental models, was quantified by real-time PCR and normalized to two housekeeping genes.



Rosiglitazone, but not metformin, treatment increased expression of genes involved in triacylglycerol storage [e.g. stearyl-CoA desaturase (3.2-fold), CD36 (1.8-fold)], structural genes [e.g. alpha-1 type-1 procollagen (1.7-fold) and GLUT4 (1.5-fold)], and decreased expression of inflammation-related genes [e.g. IL-6 (0.6-fold), chemokine (C-C motif) ligand 3 (0.4-fold)], 11beta-hydroxysteroid dehydrogenase 1 (0.6-fold), and resistin (0.3-fold) (all P < 0.05).



These results suggest that the insulin-sensitizing action of rosiglitazone involves remodeling of human adipose tissue to reduce inflammation and promote lipid storage. Furthermore, we show some important differences between thiazolidinedione action in human adipose tissue and experimental models.

title: Review: Peroxisome proliferator-activated receptor gamma and adipose tissue--understanding obesity-related changes in regulation of lipid and glucose metabolism.
abstract: Adipose tissue is a metabolically dynamic organ, serving as a buffer to control fatty acid flux and a regulator of endocrine function. In obese subjects, and those with type 2 diabetes or the metabolic syndrome, adipose tissue function is altered (i.e. adipocytes display morphological differences alongside aberrant endocrine and metabolic function and low-grade inflammation).



Articles on the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipose tissue of healthy individuals and those with obesity, metabolic syndrome, or type 2 diabetes were sourced using MEDLINE (1990-2006).



Articles were assessed to provide a comprehensive overview of how PPARgamma-activating ligands improve adipose tissue function, and how this links to improvements in insulin resistance and the progression to type 2 diabetes and atherosclerosis.



PPARgamma is highly expressed in adipose tissue, where its activation with thiazolidinediones alters fat topography and adipocyte phenotype and up-regulates genes involved in fatty acid metabolism and triglyceride storage. Furthermore, PPARgamma activation is associated with potentially beneficial effects on the expression and secretion of a range of factors, including adiponectin, resistin, IL-6, TNFalpha, plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, and angiotensinogen, as well as a reduction in plasma nonesterified fatty acid supply. The effects of PPARgamma also extend to macrophages, where they suppress production of inflammatory mediators. As such, PPARgamma activation appears to have a beneficial effect on the relationship between the macrophage and adipocyte that is distorted in obesity. Thus, PPARgamma-activating ligands improve adipose tissue function and may have a role in preventing progression of insulin resistance to diabetes and endothelial dysfunction to atherosclerosis.

title: Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces chronic colonic inflammation in rats.
abstract: Recent studies have shown the implication of the peroxisome proliferator-activated receptor gamma (PPARgamma) in control of inflammation, immune and apoptotic responses during early experimental colitis. However, there is little information about the effects of these agents on colonic mucosa under chronic inflammatory conditions. In this study, we have evaluated the effects of rosiglitazone, a PPAR-gamma agonist, on the chronic injury caused by intra-colonic administration of trinitrobenzensulfonic acid (TNBS) in rats. Rosiglitazone (1 and 5mg/kg p.o.) was administered by oral gavage, 24h after TNBS instillation and daily during 2 weeks before killing the rats. Colons were removed for histological and biochemical analysis. Administration of rosiglitazone corrected the disorders in morphology associated to lesions, significantly reduced the ulceration index, the rise of myeloperoxidase (MPO) and the levels of tumour necrosis factor alpha (TNF-alpha). In addition, rosiglitazone treatment increased prostaglandin (PG)E(2) production and returned PGD(2) to basal levels. Also, reduced cyclooxygenase (COX)-2 and nuclear transcription factor NF-kappa B (NF-kappaB) p65 proteins expression. Furthermore, treatment of rats with rosiglitazone caused a significant increase of TNBS-induced apoptosis. In summary, rosiglitazone exerts protective effects in chronic experimental colitis. The anti-inflammatory effects seem to be related to impairment of neutrophil function, absence of up-regulation of TNF-alpha and decrease of nuclear NF-kappaB p65 expression. Our results also suggest that the activation of the PPARgamma pathway reduces COX-2 overexpression, returns the increased PGD(2) values to basal levels and induces a significant increase of TNBS-induced apoptosis. We conclude that rosiglitazone represents a novel approach to the treatment of ulcerative colitis.

title: Mechanism of the Anti-Inflammatory Effect of Thiazolidinediones: Relationship with the Glucocorticoid Pathway
abstract: The glucocorticoid receptor (GR) and peroxisome proliferator-activated receptors (PPARs) play important roles in both physiological and pathological conditions such as cell differentiation, lipolysis, control of glucose metabolism, immunity, and inflammation. In fact, recent studies suggest that the thiazolidinedione (TZD) class of PPAR-γ ligands, like glucocorticoids, may also be clinically beneficial in several inflammatory diseases, even if the molecular mechanisms responsible for these activities have not yet been clarified. In this study, by using a murine model of inflammation, the carrageenin-induced paw edema in mouse, we show that the anti-inflammatory activity exhibited by the PPAR-γ agonists rosiglitazone and ciglitazone is reversed by the GR antagonist RU486 (17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one). Moreover, by using a conditional GR null cell line, we demonstrate, for the first time to our knowledge, that one of the possible mechanisms explaining the anti-inflammatory activity of TZDs is their ability to activate GR nuclear translocation. In addition, by using J774 cell line lacking PPAR-γ, we demonstrate that PPAR-γ expression could not be essential for TZD-mediated GR nuclear translocation, thus explaining, at least in part, the molecular mechanism underlying their anti-inflammatory activity.

title: Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.
abstract: Insulin resistance arises from the inability of insulin to act normally in regulating nutrient metabolism in peripheral tissues. Increasing evidence from human population studies and animal research has established correlative as well as causative links between chronic inflammation and insulin resistance. However, the underlying molecular pathways are largely unknown. In this report, we show that many inflammation and macrophage-specific genes are dramatically upregulated in white adipose tissue (WAT) in mouse models of genetic and high-fat diet-induced obesity (DIO). The upregulation is progressively increased in WAT of mice with DIO and precedes a dramatic increase in circulating-insulin level. Upon treatment with rosiglitazone, an insulin-sensitizing drug, these macrophage-originated genes are downregulated. Histologically, there is evidence of significant infiltration of macrophages, but not neutrophils and lymphocytes, into WAT of obese mice, with signs of adipocyte lipolysis and formation of multinucleate giant cells. These data suggest that macrophages in WAT play an active role in morbid obesity and that macrophage-related inflammatory activities may contribute to the pathogenesis of obesity-induced insulin resistance. We propose that obesity-related insulin resistance is, at least in part, a chronic inflammatory disease initiated in adipose tissue.

title: Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones.
abstract: PPAR gamma is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPAR gamma in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPAR gamma-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPAR gamma following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPAR gamma in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

title: Distribution of peroxisome proliferator-activated receptors (PPARs) in human skeletal muscle and adipose tissue: relation to insulin action
abstract: To evaluate the tissue distribution and possible role of the peroxisome proliferator-activated receptors (PPARs) in insulin action in fat and muscle biopsy specimens from lean, obese and subjects with Type II (non-insulin-dependent) diabetes mellitus.¶Methods. We measured PPARα, PPARβ(δ) and PPARγ protein expression by western blot analysis. The PPARγ protein was also measured in muscle before and after 3-h hyperinsulinaemic (300 mU · m–2· min–1) euglycaemic clamps.¶Results. The PPARα protein was expressed preferentially in muscle relative to fat (more than sevenfold). The PPARβ protein was similar in fat and muscle. The amount of PPARγ protein found in muscle was, on average, two-thirds of that present in fat. There was no statistically significant difference between non-diabetic and diabetic subjects in baseline (pre-clamp) muscle PPAR (α, β or γ) protein expression. Subgroup analysis showed, however, significantly higher PPARγ protein in the most insulin resistant diabetic subjects with glucose disposal rates of 3–6 mg · kg–1· min–1 compared with their age and weight matched counterparts with glucose disposal rates of 6–9 (147 ± 23 vs 88 ± 10 AU/μg protein, p≤ 0.01 in diabetic and vs 94 ± 15, p≤ 0.04 in non-diabetic subjects). Muscle PPARγ protein and glucose disposal rates were inversely correlated in diabetic subjects (r = –0.47, p≤ 0.05).¶Conclusion/interpretation. All PPARs (α, β or γ) are present in skeletal muscle and adipose tissue with different relative distributions. The PPARγ protein is abundant in skeletal muscle as well as adipose tissue. The altered expression of skeletal muscle PPARγ is consistent with a role for this nuclear protein in the impaired insulin action of Type II diabetes.

title: Muscle-specific Pparg deletion causes insulin resistance
abstract: Thiazolidinediones (TZDs) are insulin-sensitizing drugs and are potent agonists of the nuclear peroxisome proliferator-activated receptor-γ (PPAR-γ). Although muscle is the major organ responsible for insulin-stimulated glucose disposal, PPAR-γ is more highly expressed in adipose tissue than in muscle. To address this issue, we used the Cre-loxP system to knock out Pparg, the gene encoding PPAR-γ, in mouse skeletal muscle. As early as 4 months of age, mice with targeted disruption of PPAR-γ in muscle showed glucose intolerance and progressive insulin resistance. Using the hyperinsulinemic-euglycemic clamp technique, the in vivo insulin-stimulated glucose disposal rate (IS-GDR) was reduced by ∼80% and was unchanged by 3 weeks of TZD treatment. These effects reveal a crucial role for muscle PPAR-γ in the maintenance of skeletal muscle insulin action, the etiology of insulin resistance and the action of TZDs.

title: PPAR‐γ expression modulates insulin sensitivity in C2C12 skeletal muscle cells
abstract: Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) expression is very low in skeletal muscle cells, which is one of the most important target tissues for insulin and plays a predominant role in glucose homeostasis. It has recently been shown that muscle‐specific PPAR‐γ deletion in mouse causes insulin resistance. However, it is likely that the observed effects might be due to secondary interaction in whole animal. The aim of the study was to explore the role of muscle PPAR‐γ in insulin sensitivity. We stably transfected C2C12 skeletal muscle cells with plasmids containing sense or antisense constructs of PPAR‐γ and examined the effect of modulation of PPAR‐γ expression in terms of glucose uptake. Effect was also examined in insulin‐resistant C2C12 skeletal muscle cells. In transfected C2C12 cell line, the inhibition of PPAR‐γ expression (23.0±0.005%) was observed to induce insulin resistance as determined by functional assessment of 2‐deoxyglucose incorporation. Overexpression of PPAR‐γ (28.5±0.008%) produced an additional effect on insulin (100 nM) and Pioglitazone (50 μM), resulting in 42.7±3.5% increase in glucose uptake as against 29.2±2.8% in wild‐type C2C12 skeletal muscle cells differentiated under normal (2% horse serum) condition. Under similar treatment, PPAR‐γ overexpressing cells resistant to insulin exhibited enhanced glucose uptake upto 60.7±4.08%, as compared to 23.8±5.1% observed in wild‐type C2C12 skeletal muscle cells. These data demonstrate a direct involvement of PPAR‐γ in insulin sensitization of TZD action on skeletal muscle cells, and suggest that pharmacological overexpression of muscle PPAR‐γ gene in skeletal muscle might be a useful strategy for the treatment of insulin resistance.

title: Selective activation of PPARgamma in skeletal muscle induces endogenous production of adiponectin and protects mice from diet-induced insulin resistance.
abstract: The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma plays a key role in regulating whole body glucose homeostasis and insulin sensitivity. Although it is expressed most highly in adipose, it is also present at lower levels in many tissues, including skeletal muscle. The role muscle PPARgamma plays in metabolic regulation and in mediating the antidiabetic effects of the thiazolidinediones is not understood. The goal of this work was to examine the molecular and physiological effects of PPARgamma activation in muscle cells. We found that pharmacological activation of PPARgamma in primary cultured myocytes, and genetic activation of muscle PPARgamma in muscle tissue of transgenic mice, induced the production of adiponectin directly from muscle cells. This muscle-produced adiponectin was functional and capable of stimulating adiponectin signaling in myocytes. In addition, elevated skeletal muscle PPARgamma activity in transgenic mice provided a significant protection from high-fat diet-induced insulin resistance and associated changes in muscle phenotype, including reduced myocyte lipid content and an increase in the proportion of oxidative muscle fiber types. Our findings demonstrate that PPARgamma activation in skeletal muscle can have a significant protective effect on whole body glucose homeostasis and insulin resistance and that myocytes can produce and secrete functional adiponectin in a PPARgamma-dependent manner. We propose that activation of PPARgamma in myocytes induces a local production of adiponectin that acts on muscle tissue to improve insulin sensitivity.

title: Muscle-specific PPARgamma-deficient mice develop increased adiposity and insulin resistance but respond to thiazolidinediones.
abstract: Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by thiazolidinediones (TZDs) improves insulin resistance by increasing insulin-stimulated glucose disposal in skeletal muscle. It remains debatable whether the effect of TZDs on muscle is direct or indirect via adipose tissue. We therefore generated mice with muscle-specific PPARgamma knockout (MuPPARgammaKO) using Cre/loxP recombination. Interestingly, MuPPARgammaKO mice developed excess adiposity despite reduced dietary intake. Although insulin-stimulated glucose uptake in muscle was not impaired, MuPPARgammaKO mice had whole-body insulin resistance with a 36% reduction (P < 0.05) in the glucose infusion rate required to maintain euglycemia during hyperinsulinemic clamp, primarily due to dramatic impairment in hepatic insulin action. When placed on a high-fat diet, MuPPARgammaKO mice developed hyperinsulinemia and impaired glucose homeostasis identical to controls. Simultaneous treatment with TZD ameliorated these high fat-induced defects in MuPPARgammaKO mice to a degree identical to controls. There was also altered expression of several lipid metabolism genes in the muscle of MuPPARgammaKO mice. Thus, muscle PPARgamma is not required for the antidiabetic effects of TZDs, but has a hitherto unsuspected role for maintenance of normal adiposity, whole-body insulin sensitivity, and hepatic insulin action. The tissue crosstalk mediating these effects is perhaps due to altered lipid metabolism in muscle.

title: Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones.
abstract: Troglitazone, a thiazolidinedione class of antidiabetic agent, causes serious idiosyncratic hepatotoxicity. Troglitazone is metabolized to a reactive metabolite that covalently binds to cellular macromolecules, but the role of the covalent adduct in the hepatotoxicity is controversial. Because troglitazone has been found to cause cytotoxicity to hepatocytes along with mitochondrial dysfunction, we investigated the effects of troglitazone and other thiazolidinediones on mitochondrial function by using liver mitochondria fraction isolated from male CD-1 mice. Incubation of energized mitochondria with succinate in the presence of Ca2+ and troglitazone induced mitochondrial swelling, and the swelling was partially inhibited by cyclosporin A. Troglitazone also induced decreases in mitochondrial membrane potential and mitochondrial Ca2+ accumulation. These results demonstrate that troglitazone induces mitochondrial permeability transition (MPT). Similar results were obtained for ciglitazone, whereas rosiglitazone and pioglitazone, which are less hepatotoxic than troglitazone, had little effect on these mitochondria functions. It is therefore possible that the troglitazone-induced opening of MPT pore, which is not induced by rosiglitazone or pioglitazone, may contribute to the hepatotoxicity induced specifically by troglitazone.

title: Gene Expression Analysis of Troglitazone Reveals Its Impact on Multiple Pathways in Cell Culture: A Case for In Vitro Platforms Combined with Gene Expression Analysis for Early (Idiosyncratic) Toxicity Screening
abstract: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists of the thiazolidinedione family are used for the treatment of type 2 diabetes mellitus due to their ability to reduce glucose and lipid levels in patients with this disease. Three thiazolidinediones that were approved for treatment are Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). Troglitazone was withdrawn from the market due to idiosyncratic drug toxicity. Rosiglitazone and pioglitazone are still on the market for the treatment of type 2 diabetes. The authors present data from a gene expression screen that compares the impact these three compounds have in rats, in rat hepatocytes, and in the clone 9 rat liver cell line. The authors monitored the changes in expression in multiple genes, including those related to xenobiotic metabolism, proliferation, DNA damage, oxidative stress, apoptosis, and inflammation. Compared to the other two compounds, troglitazone had a significant impact on many of the pathways monitored in vitro although no major perturbation was detected in vivo. The changes detected predict not only general toxicity but potential mechanisms of toxicity. Based on gene expression analysis, the authors propose there is not just one but multiple ways troglitazone could be toxic, depending on a patient’s environment and genetic makeup, including immune response-related toxicity.

title: Troglitazone Induces Expression of PPARγ in Liver
abstract: Troglitazone is an insulin sensitizer which affects a number of target tissues. It is believed to exert these effects primarily by binding to and activating the y-isoform of peroxisome proliferator-activated receptor (PPARgamma), which in turn regulates the expression of specific genes. However, in a number of target organs, such as liver, the levels of PPARgamma are low and other isoforms predominate. In the present study, we examined whether troglitazone induces the expression of PPARgamma, thereby sensitizing cells for the action of this drug. Treatment of isolated rat hepatocytes with troglitazone induced both the mRNA and protein levels of PPARgamma in a dose-dependent fashion, with maximal levels of induction being three- to fourfold. This induction was also observed using the 15-deoxy-delta12,14-prostaglandin J2, a known natural ligand for PPARgamma, whereas ligands specific for PPARalpha were without effect. The induction of PPARgamma expression by troglitazone was also observed in livers from rats fed a diet containing troglitazone. Troglitazone had no effect on the expression of the alpha- or beta-isoforms of PPAR, the more predominant liver isoforms. These results indicate that troglitazone produces a reprogramming of PPAR isoform content in liver, which may in part underlie the mechanism whereby troglitazone sensitizes the liver to the action of insulin and/or ameliorates hyperglycemia.

title: Species-specific toxicity of diclofenac and troglitazone in primary human and rat hepatocytes.
abstract: Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.

title: Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes
abstract: 1. Troglitazonewas the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 muM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = ~0.8 muM) and dexamethasone (40-50 muM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.

title: PPARγ2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes
abstract: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.

title: Liver cell models in in vitro toxicology.
abstract: In vitro liver preparations are increasingly used for the study of hepatotoxicity of chemicals. In recent years their actual advantages and limitations have been better defined. The cell models, slices, and mainly primary hepatocyte cultures, appear to be the most powerful in vitro systems, as liver-specific functions and responsiveness to inducers are retained either for a few days or several weeks depending on culture conditions. Maintenance of phase I and phase II xenobiotic metabolizing enzyme activities allows various chemical investigations to be performed, including determination of kinetic parameters, metabolic profile, interspecies comparison, inhibition and induction effects, and drug-drug interactions. In vitro liver cell models also have various applications in toxicology: screening of cytotoxic and genotoxic compounds, evaluation of chemoprotective agents, and determination of characteristic liver lesions and associated biochemical mechanisms induced by toxic compounds. Extrapolation of the results to the in vivo situation remains a matter of debate. Presently, the most convincing applications of liver cell models are the studies on different aspects of metabolism and mechanisms of toxicity. For the future, there is a need for better culture conditions and differentiated hepatocyte cell lines to overcome the limited availability of human liver tissues. In addition, strategies for in vitro analysis of potentially toxic chemicals must be better defined.

title: Transdifferentiation of hepatocyte‐like cells from the human hepatoma HepaRG cell line through bipotent progenitor
abstract: Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte‐like and biliary‐like cells at confluence. If hepatocyte‐like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21CIP1 and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of β‐catenin localization and HNF3β expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells.  Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors. (HEPATOLOGY 2007;45:957–967.

title: Evolving concepts in liver tissue modeling and implications for in vitro toxicology.
abstract: The development of human cell models stably expressing functional properties of the in vivo cells they are derived from for predicting toxicity of chemicals is a major challenge. For mimicking the liver, a major target of toxic chemicals, primary hepatocytes represent the most pertinent model. Their use is limited by interdonor functional variability and early phenotypic changes although their lifespan can be extended not only by culturing in a 2D dimension under sophisticated conditions but also by the use of synthetic and natural scaffolds as 3D supporting templates that allow cells to have a more stable microenvironment. Hepatocytes derived from stem cells could be the most appropriate alternative but up to now only liver progenitors/hepatoblasts are obtained in vitro. A few hepatocyte cell lines have retained a variable set of liver-specific functions. Among them are the human hepatoma HepaRG cells that express drug metabolism capacity at levels close to those found in primary hepatocytes making them a suitable model for both acute and chronic toxicity studies. New screening strategies are now proposed based on miniaturized and automated systems; they include the use of microfluidic chips and cell chips coupled with high content imaging analysis. Toxicogenomics technologies (particularly toxicotranscriptomics) have emerged as promising in vitro approaches for better identification and discrimination of cellular responses to chemicals. They should allow to discriminate compounds on the basis of the identification of a set of markers and/specific signaling pathways.

title: EXPRESSION OF CYTOCHROMES P450, CONJUGATING ENZYMES AND NUCLEAR RECEPTORS IN HUMAN HEPATOMA HepaRG CELLS
abstract: Most human hepatocyte cell lines lack a substantial set of liver-specific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor α), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.

title: Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.
abstract: Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.

title: The role of conjugation in hepatotoxicity of troglitazone in human and porcine hepatocyte cultures.
abstract: In primary human and porcine hepatocyte cultures, we investigated the relationship between metabolism and cytotoxicity of troglitazone. Treatment of human hepatocytes for 2 h with 10, 20, 25, 35, and 50 microM troglitazone in protein-free medium resulted in concentration-dependent decreases in total protein synthesis. Decreases at 10 and 20 microM were reversible by 24 h, however protein synthesis did not recover at concentrations >/=25 microM. Troglitazone at 50 microM caused cellular death. In porcine hepatocytes, 100 microM troglitazone was lethal, whereas at 50 microM, protein synthesis completely recovered by 24 h. Recovery in protein synthesis was associated with metabolism of parent drug, whereas toxicity correlated (r(2) = 0.82) with accumulation of unmetabolized troglitazone. By 1 h, in human hepatocytes, troglitazone was metabolized to similar amounts of sulfate and quinone metabolites with little glucuronide detected. In contrast, porcine hepatocytes metabolized troglitazone to the similar amounts of glucuronide and the quinone metabolites with little sulfate detected. Exposure of human hepatocytes to a combination of 10 microM troglitazone and 10 microM 2,4-dichloro-4-nitrophenol resulted in a 70% decrease in protein synthesis, associated with 90% inhibition in the formation of troglitazone sulfate, a 4-fold increase in unmetabolized troglitazone, and no effect on formation of the quinone metabolite. Treatment with a combination of acetaminophen or phenobarbital with 20 microM troglitazone resulted in sustained decrease in protein synthesis associated with inhibition of sulfation and accumulation of troglitazone. These results suggest that inhibition of troglitazone sulfation may result in increased hepatotoxicity due to exposure to parent drug, or increased metabolism by alternate pathways.

title: Increased sensitivity for troglitazone-induced cytotoxicity using a human in vitro co-culture model.
abstract: Drug-induced hepatotoxicity is a major reason for withdrawal of drugs from development as well as from the market. A major problem predicting hepatotoxicity is the lack of suitable predictive in vitro system. Drug-induced hepatotoxicity is usually associated with the recruitment of immune cells to the liver accelerating an inflammatory response often initiated by activation of the Kupffer cells. In order to evaluate whether the introduction of inflammatory cells could increase the sensitivity for drug-induced cytotoxicity we developed an in vitro co-culture system based on two human cell lines; a hepatoma (Huh-7) and monocytic (THP-1) cell line. As model drugs we chose two peroxisome proliferator activated receptor gamma (PPAR gamma) agonists, the hepatotoxic troglitazone and the non-hepatotoxic rosiglitazone. In the co-cultures, troglitazone caused an enhanced cytotoxicity as compared to single cultures of either cell line, whereas little cytotoxicity was seen after treatment with rosiglitazone. Troglitazone treatment increased gene expression of pro-inflammatory mediators and stress-related genes in both cell types, which in general was more pronounced in co-cultures than in single cell cultures. Based on these results we suggest that co-cultures of human hepatoma cells and monocytes might provide an important in vitro system for better prediction of cytotoxicity mediated by potential hepatotoxins.

title: Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.
abstract: Troglitazone (TRO), an effective thiazolidinedione antidiabetic agent, was reported to produce idiosyncratic hepatotoxic effects in some individuals. In contrast, rosiglitazone (RSG), in the same group of agents, has no significant toxic effects and now is widely used. In this study, human hepatoma (HepG2) cell lines were exposed to various doses of TRO as well as RSG (0, 25, 50, and 75 microM) for 48 h. Cell lysates were separated by two-dimensional electrophoresis, and the gels were stained with coomassie brilliant blue to compare the spot profiles. The greatest protein expression at a MW of 75 kDa and isoelectric point of 5 was specifically increased with TRO treatments of 50 and 75 microM. The spot was identified as a mixture of immunoglobulin heavy chain binding protein (BiP) and, to a lesser extent, protein disulfide isomerase-related protein (PDIrp). Immunoblot analyses showed that the BiP protein was dose-dependently increased by TRO treatment and, to a lower degree, by RSG. These effects were also correlated with the high induction of BiP mRNA by TRO (50 and 75 microM) and the lower induction by RSG. However, both treatments showed no significant effects on PDIrp expression. The toxic effects of TRO in relation to the overexpression of BiP were also demonstrated in HLE cells, another human hepatoma cell line. In HLE cells, the inhibition of BiP expression by small interference RNA rendered cells more susceptible to the toxic effects of TRO. These results suggest that the overexpression of BiP is a defense mechanism of the endoplasmic reticulum in response to TRO-induced toxicity.

title: Peroxisome proliferator-activated receptor gamma ligands inhibit cell growth and induce apoptosis in human liver cancer BEL-7402 cells.
abstract: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.



The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examined in BEL-7402 liver cancer cells. Apoptosis was characterized by Hochest33258 staining, DNA fragmentation, TUNEL and ELISA, and cell cycle kinetics by FACS. Modulation of apoptosis related caspases expression by PPAR gamma ligands was examined by Western blot.



PPARgamma ligands, 15-deoxy-(12), (14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of BEL-7402 cells. Both 15d-PGJ2 and TGZ induced BEL-7402 cell death in a dose dependent manner, which was associated with an increase in fragmented DNA and TUNEL-positive cells. At concentrations of 10 and 30 microM, 15d-PGJ(2) or troglitazone increased the proportion of cells with G(0)/G(1) phase DNA content and decreased those with S phase DNA content. There was no significant change in the proportion of cells with G(2)/M DNA content. The activities of Caspases-3, -6, -7 and -9 were increased by 15d-PGJ2 and TGZ treatment, while the activity of Caspase 8 had not significantly changed.



The present results suggest the potential usefulness of PPAR gamma ligands for chemoprevention and treatment of liver cancers.

title: Troglitazone but not rosiglitazone induces G1 cell cycle arrest and apoptosis in human and rat hepatoma cell lines.
abstract: Rosiglitazone (RSG), an agonist of peroxisome proliferator-activated receptor gamma (PPARgamma), induces minor toxicity in humans relative to another PPARgamma agonist, troglitazone (TRO). In contrast, recent reports suggest that RSG causes growth arrest and apoptosis of normal and cancerous cells. Therefore, in this study, we investigated the relative toxicities of TRO and RSG on three different hepatoma cell lines, and observed that TRO, but not RSG, was cytotoxic. Additionally, we studied the mechanism by which TRO induced damage to HepG2 hepatoma cells. Our results indicated that TRO increased the levels of p53, p27, and p21, while it reduced the levels of cyclin D1 and phospho-Rb in a time-dependent manner. Increased p27 and p21 levels coincided with reduced activities of cell cycle dependent kinases (cdk) such as cdk2- and cyclin A-protein kinases 24 h after TRO treatment. These results demonstrate that TRO, but not RSG, causes G1 arrest of hepatoma cells, most likely through changing the levels of cell cycle regulators. Furthermore, because RSG did not affect the levels of cell cycle regulators, TRO-mediated growth inhibition appears independent of PPARgamma activation.

title: Involvement of p21WAF1/Cip1, p27Kip1, and p18INK4c in troglitazone‐induced cell‐cycle arrest in human hepatoma cell lines
abstract: Peroxisome proliferator–activated receptor γ (PPARγ) regulates cell growth and differentiation. Recent evidence has suggested that PPARγ ligands had anti‐tumor effects through inhibiting cell growth and inducing cell differentiation in several types of malignant neoplasm. In the present study, we investigated: 1) the expression of PPARγ in both human hepatoma cell lines and 5 resected human hepatocellular carcinoma (HCC) tissues; 2) the growth‐inhibitory effect of troglitazone, a PPARγ ligand, on those hepatoma cells; and 3) the molecular mechanisms of troglitazone‐induced cell‐cycle arrest. Five hepatoma cell lines, HLF, HuH‐7, HAK‐1A, HAK‐1B, and HAK‐5, were used. The mRNA expression levels of PPARγ, p21WAF1/Cip1, and p27Kip1 were determined by real‐time quantitative reverse transcription‐polymerase chain reaction. The expression of cell cycle–regulating proteins, such as p21, p27, p18INK4c, cyclin E, and pRb, was examined using Western blotting. PPARγ was constitutively expressed in all the cell lines and the HCC tissues used in this study. A cytostatic effect of troglitazone was found in those cell lines, and this inhibition of cell growth was dosage‐dependent. G0/G1 arrest was apparently demonstrated in flow cytometric analysis in HLF, HAK‐1A, HAK‐1B, and HAK‐5, all of which showed an increased expression of p21 protein. However, HuH‐7, lacking p21 protein expression, did not demonstrate clear arrest in the cell‐cycle analysis. HLF, which was deficient in the protein product of the retinoblastoma tumor‐suppressor gene (pRb), responded most profoundly to troglitazone, showing an increased expression in not only p21, but also in p27 and in p18. These findings suggested that p21, p27, and p18 might be involved in troglitazone‐induced cell‐cycle arrest in human hepatoma cells.

title: Troglitazone induces p27Kip1‐associated cell‐cycle arrest through down‐regulating Skp2 in human hepatoma cells
abstract: Increasing evidence has confirmed that ligands for peroxisome proliferator‐activated receptor γ (PPARγ) exhibit antitumoral effects through inhibition of cell proliferation and induction of cell differentiation in several malignant neoplasms. Recently, we have documented the accumulation of a cyclin‐dependent kinase inhibitor, p27Kip1, as well as an unexpected accumulation in cyclin E in G1‐arrested human hepatoma cells treated with the PPARγ ligand troglitazone. Simultaneous accumulations in both p27Kip1 and cyclin E are known to be characteristic phenotypes in cells derived from mice lacking Skp2, an F‐box protein component of the SCF ubiquitin‐ligase complex. Thus, the aim of the present study was to assess whether Skp2 might be involved in the down‐regulation of p27Kip1 in troglitazone‐treated human hepatoma cells. A striking decrease in Skp2 expression and a reciprocal increase in p27Kip1 expression were found in troglitazone‐treated hepatoma cells but not in those cells treated with other PPARγ ligands such as pioglitazone and ciglitazone. Quantitative real‐time RT‐PCR analysis showed that troglitazone down‐regulated Skp2 at the mRNA levels. Consistently, ectopic overexpression in Skp2 brought resistance to troglitazone, resulting in a decreased population of arrested cells at the G1 phase compared with that in the mock‐transfected cells. In surgically resected hepatocellular carcinoma (HCC) tissue, an increased expression in Skp2 was found in both the moderately differentiated HCCs and the poorly differentiated HCCs. In conclusion, troglitazone attenuated Skp2 expression, thereby promoting p27Kip1 accumulation in human hepatoma cells. This therapeutic potential of the ligand may lead to new cell‐cycle‐based antitumor strategies for advanced HCCs.

title: Dose- and time-dependent effects of phenobarbital on gene expression profiling in human hepatoma HepaRG cells.
abstract: Phenobarbital (PB) induces or represses a wide spectrum of genes in rodent liver. Much less is known about its effects in human liver. We used pangenomic cDNA microarrays to analyze concentration- and time-dependent gene expression profile changes induced by PB in the well-differentiated human HepaRG cell line. Changes in gene expression profiles clustered at specific concentration ranges and treatment times. The number of correctly annotated genes significantly modulated by at least three different PB concentration ranges (spanning 0.5 to 3.2 mM) at 20 h exposure amounted to 77 and 128 genes (p< or =0.01) at 2- and 1.8-fold filter changes, respectively. At low concentrations (0.5 and 1 mM), PB-responsive genes included the well-recognized CAR- and PXR-dependent responsive cytochromes P450 (CYP2B6, CYP3A4), sulfotransferase 2A1 and plasma transporters (ABCB1, ABCC2), as well as a number of genes critically involved in various metabolic pathways, including lipid (CYP4A11, CYP4F3), vitamin D (CYP24A1) and bile (CYP7A1 and CYP8B1) metabolism. At concentrations of 3.2 mM or higher after 20 h, and especially 48 h, increased cytotoxic effects were associated with disregulation of numerous genes related to oxidative stress, DNA repair and apoptosis. Primary human hepatocyte cultures were also exposed to 1 and 3.2 mM PB for 20 h and the changes were comparable to those found in HepaRG cells treated under the same conditions. Taken altogether, our data provide further evidence that HepaRG cells closely resemble primary human hepatocytes and provide new information on the effects of PB in human liver. These data also emphasize the importance of investigating dose- and time-dependent effects of chemicals when using toxicogenomic approaches.

title: PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma
abstract: BackgroundAlthough PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents.MethodsWe have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2).Results15-deoxy-prostaglandin J2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis.ConclusionTroglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent.

title: Signaling pathways involved in induction of GADD45 gene expression and apoptosis by troglitazone in human MCF-7 breast carcinoma cells
abstract: We previously reported that the PPARγ agonist troglitazone (TRO) inhibits proliferation and induces apoptosis in human MCF-7 breast carcinoma cells. To understand the mechanisms of antiproliferative and pro-apoptotic effects of TRO, we screened a limited DNA array containing 23 genes involved in regulating either the cell cycle and/or apoptosis. Four of the 23 genes screened exhibited regulation by TRO, with growth arrest and DNA damage-inducible gene 45 (GADD45) being the most strongly upregulated. TRO induced GADD45 mRNA expression in a time- and dose-dependent manner. Depletion of GADD45 by siRNA abrogated TRO-induced apoptosis in MCF-7 cells demonstrating the physiological relevance of GADD45 upregulation. Signaling pathways mediating TRO-induced GADD45 were also investigated. Several mitogen-activated protein kinase (MAPK) pathways were involved in the induction of GADD45 by TRO. Inhibition of the c-jun N-terminal kinase MAPK pathway by SP600125 partially abolished TRO-induced GADD45 mRNA, and protein expression and apoptosis. In contrast, inhibition of the p38 MAPK pathway by SB203580, or through overexpression of a dominant-negative mutant of p38 MAPK, augmented GADD45 mRNA induction and GADD45 promoter activation as well as cell apoptosis by TRO. Blockade of the extracellular signal-regulated kinase MAPK pathway by PD98059 also enhanced TRO's effects on GADD45 and apoptosis. Two other PPARγ agonists pioglitazone and rosiglitazone did not induce GADD45 expression. Our finding of GADD45 induction by TRO may provide a new insight concerning the mechanisms for TRO's antiproliferative and pro-apoptotic effects in breast cancer cells.

title: Activation of peroxisome proliferator-activated receptor-γ stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells
abstract: (2002) 21, 8220–8221.  The authors wish to apologise that the names of the fifth and last authors have been spelled incorrectly. The correct spelling is given above.

title: Applications of microarrays with toxicologically relevant genes (tox genes) for the evaluation of chemical toxicants in Sprague Dawley rats in vivo and human hepatocytes in vitro.
abstract: Microarrays with toxicologically relevant genes (tox genes) have been developed in our laboratory for toxicogenomics studies in rat, dog and man. The genes were chosen using published information as well as a discovery process for genes responsive to toxic treatments using transcription profiling experiments conducted with rats and dogs. In addition to published information human tox genes were derived from rat tox genes based on gene homology. Using the microarray with rat-specific tox genes, a database containing gene expression, histopathology, and clinical chemistry findings has been generated for 89 compounds. Analysis of the database indicates that treatment with toxic compounds induces specific gene expression patterns. Dose- and time-dependent response relationships in gene expression were observed for treatment with toxic compounds. Gene expression at 24h was found to correlate well with organ toxicity observed at 72 h. Mining of the database led to the selection of specific groups of genes (predictive gene sets) whose expression patterns are predictive of organ toxicity with a high degree of accuracy (approximately 90%). The data also provide insight on toxic mechanism and gene regulation pathways. For instance, carbon tetrachloride and chloroform treatments were found to decrease the expression of the cytochrome P450 isoform 3A1 gene while enhancing the expression of the multiple drug resistance gene MDR1 in liver, clearly demonstrating that the CYP3A1 and MDR1 genes were not co-regulated as postulated by some researchers. This approach, the use of gene expression as an endpoint to define organ toxicity, is extended to the definition of human drug toxicity using primary human hepatocytes as a test system. Preliminary results demonstrate that the toxic drug, troglitazone, can be clearly distinguished from the less toxic analogues, rosiglitazone and pioglitazone based on their effects on tox gene expression in human hepatocytes. Our results with both rats in vivo and human hepatocytes in vitro suggest that microarrays with toxicologically relevant genes can be used routinely for the evaluation of chemical toxicity.

title: PPARγ Ligand Troglitazone Lowers Cholesterol Synthesis in HepG2 and Caco-2 Cells via a Reduced Concentration of Nuclear SREBP-2
abstract: 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)-γ 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 μM of troglitazone, a synthetic PPARγ agonist, for 4 hrs. Incubation with 10 or 30 μM 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 μM 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 μM of troglitazone compared to control cells (P < 0.05). In conclusion, this study shows that PPARγ 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.

title: HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans
abstract: HepaRG is a highly differentiated cell line that displays several hepatocyte-like functions, including drug-metabolizing enzymes. In this study, the HepaRG cells were characterized and evaluated as an in vitro model to predict cytochrome P450 (P450) enzyme induction of drugs in humans. Exposure of HepaRG cells to prototypical inducers resulted in induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA, as well as phenacetin O-dealkylase, bupropion hydroxylase, diclofenac 4′-hydroxylase, and midazolam 1′-hydroxylase activities. The observed induction is consistent with the previously reported expression of the nuclear receptors pregnane X receptor, constitutive androstane receptor, and aryl hydrocarbon receptor, which are necessary for a P450 induction response. To avoid problems with toxicity and solubility, the induction potency of test compounds was evaluated by calculating the concentrations leading to a 2-fold increase of baseline mRNA or enzyme activity levels (F2 values) instead of EC50 values from full dose-response curves. For CYP3A4 mRNA, the obtained F2 values were related to the in vivo exposure [area under the plasma concentration versus time curve (AUC)] of the inducer (AUC/F2). This score was then correlated with the decrease in AUC for a CYP3A probe drug, administered before and after treatment with the inducing agent. By using this method an excellent correlation (R2 = 0.863) was obtained, which implies that the degree of CYP3A induction in vivo can be predicted from CYP3A4 mRNA induction in HepaRG cells. The present study shows that HepaRG cells are a valuable model to be used for prediction of induction of drug-metabolizing P450 enzymes in vivo in humans.

title: Expression profiling of interindividual variability following xenobiotic exposures in primary human hepatocyte cultures.
abstract: To examine the magnitude of human variability across the entire transcriptome after chemical challenge, we profiled gene expression responses to three different prototypic chemical inducers in primary human hepatocyte cultures from ten independent donors. Correlation between basal expression in any two hepatocyte donors ranged from r(2) values of 0.967 to 0.857, and chemical treatment tended to negatively impact correlation between donors. Including anticipated target genes, 10,812, 8373, and 7847 genes were changed in at least one donor by Aroclor 1254 (A1254), di(2-ethylhexyl) phthalate (DEHP), and phenobarbital (PB), respectively. A subset of these gene targets (n=41) were altered with a high level of reproducibility in at least 9 donors, gene responses that correlated well with literature-reported mechanism of action. Filtering responses to the level of gene subsets clarified the biological impact associated with the respective chemical effectors, in lieu of substantial interindividual variation among donor responses. In these respects, the use of hierarchical clustering methods successfully grouped seven of the ten donors into chemical-specific rather than donor-specific clusters. However, at the whole-genome level, the magnitude of conserved gene expression changes among donors was surprisingly small, with fewer than 50% of the gene responses altered by a single chemical conserved in more than one donor. The use of higher level descriptors, such as those defined by the PANTHER classification system, may enable more consistent categorization of gene expression changes across individuals, as increased reproducibility was identified using this method.

title: Clinical Pharmacokinetics of Troglitazone
abstract: Troglitazone is a new thiazolidinedione oral antidiabetic agent approved for use to improve glycaemic control in patients with type 2 diabetes. It is rapidly absorbed with an absolute bioavailability of between 40 and 50%. Food increases the absorption by 30 to 80%. The pharmacokinetics of troglitazone are linear over the clinical dosage range of 200 to 600mg once daily. The mean elimination half-life ranges from 7.6 to 24 hours, which facilitates a once daily administration regimen. The pharmacokinetics of troglitazone are similar between patients with type 2 diabetes and healthy individuals.In humans, troglitazone undergoes metabolism by sulfation, glucuronidation and oxidation to form a sulfate conjugate (M1), glucuronide conjugate (M2) and quinone metabolite (M3), respectively. M1 and M3 are the major metabolites in plasma, and M2 is a minor metabolite. Age, gender, type 2 diabetes, renal impairment, smoking and race do not appear to influence the pharmacokinetics of troglitazone and its 2 major metabolites. In patients with hepatic impairment the plasma concentrations of troglitazone, M1 and M3 increase by 30%, 4-fold, and 2-fold, respectively.Cholestyramine decreases the absorption of troglitazone by 70%. Troglitazone may enhance the activities of cytochrome P450 (CYP) 3A and/or transporter(s) thereby reducing the plasma concentrations of terfenadine, cyclosporin, atorvastatin and fexofenadine. It also reduces the plasma concentrations of the oral contraceptive hormones ethinylestradiol, norethindrone and levonorgestrel. Troglitazone does not alter the pharmacokinetics of digoxin, glibenclamide (glyburide) or paracetamol (acetaminophen). There is no pharmacodynamic interaction between troglitazone and warfarin or alcohol (ethanol).Pharmacodynamic modelling showed that improvement in fasting glucose and triglyceride levels increased with dose from 200 to 600mg. Knowledge of systemic troglitazone exposure within a dose group does not improve the prediction of glucose lowering response or adverse effects beyond those based on the administered dose.