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Published as doi: 10.1096/fj.06-6511fje.
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(The FASEB Journal. 2006;20:2609-2611.)
© 2006 FASEB

Rosiglitazone inhibits mouse liver regeneration

Yumirle P. Turmelle*, Olga Shikapwashya*, Shu Tu*, Paul W. Hruz*,{ddagger}, Qingyun Yan* and David A. Rudnick*,{dagger},1

* Department of Pediatrics,

{dagger} Department of Molecular Biology and Pharmacology, and

{ddagger} Department of Cellular Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA

1Correspondence: Department of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., Box 8208, St. Louis, MO 63110, USA. E-mail: rudnick_d{at}kids.wustl.edu

ABSTRACT

The remarkable regenerative potential of the liver is well known. Recent investigations have shown that this regenerative response is impaired in mouse models of fatty liver disease. Other studies demonstrate that mice engineered for liver-specific overexpression of the peroxisome proliferator activated receptor gamma (PPAR{gamma}) develop significant hepatic steatosis. These observations suggest that precise regulation of hepatic PPAR{gamma} activity may be essential for normal liver regeneration. To test this hypothesis, we analyzed the effects of PPAR{gamma}-activating thiazolidinediones on liver regeneration in the rodent partial hepatectomy model. Thiazolidinediones with different PPAR{gamma}-activating potencies were administered to mice, and those mice were subjected to partial hepatectomy and analyzed for resulting effects on hepatocellular proliferation and signaling pathways important during normal liver regeneration. The results showed that thiazolidinediones suppress liver regeneration with efficacies that correlate with their relative PPAR{gamma}-activating potencies. These studies provide the first evidence linking regulation of PPAR{gamma} activity and the hepatic regenerative response.—Turmelle, Y. P., Shikapwashya, O., Tu, S., Hruz, P. W., Yan, Q., Rudnick, D. A. Rosiglitazone inhibits mouse liver regeneration.


Key Words: partial hepatectomy • mouse model • cell cycle

THE HEPATIC REGENERATIVE RESPONSE is characterized by rapid proliferation of normally quiescent hepatocytes in remnant liver tissue, leading to appropriate restoration of liver mass (1 2 3 4) . The coordinated signaling events that are induced following partial hepatectomy include activation of TNF{alpha}-IL6 signaling (5 6 7) , generation of mitochondrial reactive oxygen species (ROS) (8) and prostaglandins (9) , and activation of stress- and mitogen-activated-protein kinase cascades (10) . These events promote activation of NF-{kappa}B, STAT3, AP1, and other transcription factors (11 12 13) , which direct an immediate-early gene expression program (14) culminating in growth factor-dependent hepatocellular re-entry into and progression through the cell cycle and restoration of normal hepatic mass. Subsequently, the regenerative response is terminated. Despite the knowledge gained from experimental analyses of liver regeneration, a clear and integrated understanding of the specific mechanisms responsible for initiation, propagation, and termination of the hepatic regenerative response remains elusive.

Recent studies have shown that liver regeneration is impaired in a number of animal models of fatty liver disease (15 16 17 18 19 20) . On the basis of those observations and a report describing significant hepatic fat accumulation in a liver-specific peroxisome proliferator activated receptor gamma (PPAR{gamma}) overexpressing mouse model (21) , we hypothesized that PPAR{gamma} activity is likely to be regulated during normal liver regeneration and that disruption of such regulation could impair the regenerative response. To test this hypothesis, we investigated the effects of PPAR{gamma}-activating thiazolidinediones on liver regeneration. The thiazolidinediones are a class of antidiabetic drugs originally developed for treatment of patients with insulin-resistant diabetes mellitus (22 , 23) . The specific mechanisms responsible for their insulin-sensitizing action, though incompletely understood, involve activation of PPAR{gamma} (24) . In this manuscript, we report the results of our analyses investigating the effects of thiazolidinedione administration on liver regeneration in the rodent partial hepatectomy model. Our results show that thiazolidinediones impair liver regeneration with efficacies corresponding to their relative potencies of PPAR{gamma} activation (i.e., rosiglitazone>pioglitazone>troglitazone, (22 , 25 26 27) ). These data implicate, for the first time, PPAR{gamma} signaling as an important regulatory determinant of hepatic regeneration.

MATERIALS AND METHODS

Animal husbandry and surgery
Eight to twelve week-old male C57Bl/6J mice (Jackson Laboratory, Bar Harbor, ME) were kept on 12:12-h dark-light cycles and maintained on standard mouse chow and water before and after surgery. Mice were treated with 10 mg/kg bid troglitazone (Cayman Company, Ann Arbor, MI), pioglitazone (Eli Lilly and Company, Indianapolis, IN), rosiglitazone (GlaxoSmithKline, Research Triangle Park, NC), or vehicle (made as a suspension in a 1:1 mixture of sterile PBS and polyethylene glycol, Sigma, St. Louis, MO) by gavage beginning 2 days before surgery. This dosing regimen was chosen because it is well tolerated and within the range typically used to elicit pioglitazone- and rosiglitazone-mediated PPAR{gamma}-dependent changes in insulin signaling in mouse models of diabetes (28 , 29) . Partial hepatectomy or sham surgery, recovery, and plasma and tissue harvest were performed, as described previously (9 , 30 , 31) . Briefly, mice were sedated with methoxyflurane (Medical Developments Australia, Victoria, Australia) and subjected to midventral laparotomy with exposure of the left and median hepatic lobes. This was followed by sequential ligation and resection of the median and left lobes and closure of the peritoneal and skin wounds. For sham surgeries, animals were sedated, subjected to laparotomy with exposure of hepatic lobar structures, and then closed. Hepatectomized and sham-operated animals were allowed to recover until the time of sacrifice (by inhaled carbon dioxide) and recovery of plasma and liver tissue (right lobe). Three to six animals were examined at each time point and for each treatment group or genotype (Three vehicle-, pioglitazone-, and rosiglitazone-treated animals were examined at 24 and 72 h after hepatectomy; 5–6 vehicle-, pioglitazone-, and rosiglitazone-treated animals were examined at 36 and 48 h after hepatectomy; 3 troglitazone-treated animals were examined at 24 and 36 h after hepatectomy; 3 vehicle- and rosiglitazone-treated animals were examined at 0, 2, 4, 6, and 12 h after hepatectomy; 3 pioglitazone-treated animals were examined at 6 and 12 h after hepatectomy; 3 vehicle- and rosiglitazone-treated animals were examined at 12 and 24 h after sham surgery). All experiments were approved by the Animal Studies Committee of Washington University and conducted in accordance with institutional guidelines and the criteria outlined in the "Guide for Care and Use of Laboratory Animals" (NIH publication 86–23).

Histology and immunohistochemistry
Liver histology and hepatocellular bromodeoxyuridine (BrdU) incorporation were assessed as described previously ((9 , 30 , 31) . Briefly, animals were injected with 100 mg/kg BrdU 1 h before sacrifice. After harvesting, a portion of the right hepatic lobe was fixed in formalin, paraffin-embedded, and stained either with hematoxylin and eosin or for nuclear BrdU incorporation. The frequency of nuclear BrdU labeling was determined by examination of at least three random 400 x fields and at least 300 cells and nuclei in each tissue section.

Cytokine and transaminase determination
Circulating TNF{alpha} and interleukin (IL)-6 levels were determined on plasma by ELISA (BD Biosciences, San Diego, CA), as described previously (9) . Aminotransferase activity was determined by the St. Louis Children’s Hospital Clinical Laboratory.

Immunoblot analysis
Whole cell lysates were made from snap frozen liver, and their protein concentration was determined as described previously (9) . Twenty-five-microgram aliquots of protein lysate were subjected to SDS-PAGE, followed by electrophoretic transfer to nitrocellulose. Filters were probed with primary antibody (Ab) (STAT3, phospho-STAT3, p42/44, phospho-p42/44, p38, phospho-p38, cyclin B1, p27Kip, Cell Signaling Technology, Beverly, MA; cyclin D1, Upstate, Lake Placid, NY; p21Waf1/Cip1 Santa Cruz Biotechnology, Santa Cruz, CA) followed by a horseradish peroxidase-conjugated secondary Ab, and then developed using the enhanced chemiluminescence (ECL) system (Amersham, Piscataway, NJ). Densitometric analysis was performed with Scion Image data analysis software (Scion Corp., Frederick, MD).

Real-time reverse transcriptase-polymerase chain reaction-based gene expression analysis
Total RNA was prepared from mouse liver tissue, reverse-transcribed to cDNA, and analyzed for levels of expression of specific genes of interest using real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR), as described previously (30) . For each gene analyzed, an aliquot of cDNA was added to a reaction mixture containing gene-specific forward and reverse primers (cyclin D1, forward primer: 5'-GAAGGAGACCATTCCCTTGA-3', reverse primer: 5'-GTTCACCAGAAGCAGTTCCA-3'; cyclin B1, forward primer: 5'-AATCCTTGCAGTGAGTGACG-3', reverse primer: 5'-CCAGTTGTCGG-AGATAAGCA-3'; SOCS3, forward primer: 5'-TTCAGCTCCAAAAGCGAGTA-3', reverse primer: 5'-GCTCCAGTAGAATCCGCTCT-3'; C/EBP{alpha}, forward primer: 5'-CCTGAGAGCTCCTTGGTCA-3', reverse primer: 5'-GAAACCATCCTCTGGGTC-TC-3'; C/EBPß, forward primer: 5'-ACGACTTCCTCTCCGACCT-3', reverse primer: 5'-GAG-GCTCACGTAACCGTAGTC-3'; C/EBP{delta}, forward primer: 5'-GACCTCTTCAACAGCAACCA-3', reverse primer: 5'-CTAGCGACAGACCCCACAC-3'; p21Waf1/Cip1 forward primer: 5'-GGGTAAACAGGACGGTGACT-3', reverse primer: 5'-TTCCACCACACCATGAGACT-3'; p27Kip1 forward primer: 5'-TCTCAGGCAAACTCTGAGGA-3', reverse primer: 5'-CTTCCTCATCCCTGGACACT-3'; Skp2 forward primer: 5'-GCGCTAAAACAGGAGTCTGG-3', reverse primer: 5'-CCTATGCATGGTGTCCACTG-3'), deoxy-nucleotides, TaqDNA polymerase, and SYBR (Bio-Rad, Hercules, CA). Quantification of cDNA was based on monitoring increased SYBR fluorescence during exponential phase amplification in a real-time PCR machine (Bio-Rad) and determination of the PCR cycle number at which the amplified product exceeded a defined threshold (the "crossing threshold"). These data were also standardized to the expression of ß2-microglobulin, which has commonly been used as a reference gene in analyses of regulation of gene expression during liver regeneration (6 , 7 , 32 , 33) and exhibits an expression pattern very similar to other genes also used for this purpose (e.g., ß-actin (34) and glyceraldehyde-3-phosphate dehydrogenase (35) , data not shown). These standardized data were used to calculate fold differences in gene expression. Specificity of this assay was verified for each gene under analysis by confirmation of predicted product size and uniformity using melt curves and agarose-electrophoresis of the PCR products. Specificity was further confirmed by simultaneous analysis of a "reverse-transcribed" reaction mixture containing all components except reverse transcriptase.

Statistical analysis
Data were analyzed using SigmaPlot and SigmaStat software (SPSS, Inc., Chicago, IL). Unpaired Student’s t test for pair-wise comparisons and ANOVA for multiple groups (with the Holm-Sidak and Dunn’s method for post hoc comparison) were used to determine statistical significance of differences in hepatocellular BrdU incorporation, mitotic body frequency, hepatic gene and protein expression, and cytokine and aminotransferase levels. Kaplan-Meier survival analysis was performed using the LogRank statistic to compare outcomes between treatment groups. Data are reported as means ± SE.

RESULTS

Thiazolidinediones inhibit hepatocellular proliferation and cyclin expression during liver regeneration with efficacies corresponding to their relative potencies of PPAR{gamma} activation
To begin to characterize the effects of PPAR{gamma}-activating thiazolidinediones on liver regeneration, hepatocellular proliferation was determined in C57BL/6J mice treated with troglitazone, pioglitazone, or rosiglitazone or with vehicle control 36 h after partial hepatectomy. Rosiglitazone is a more potent PPAR{gamma} activator than pioglitazone, and pioglitazone is more potent than troglitazone (22 , 25 26 27) . Each of these drugs was administered at a dose of 10 mg/kg bid, which is well tolerated and in the range typically used to elicit PPAR{gamma}-dependent changes in animal models (28 , 29) . The 36-h time point corresponds to peak proliferation in C57BL/6J untreated mice (9) . This analysis showed that hepatocellular proliferation is significantly reduced by treatment with rosiglitazone (6±3% hepatocellular BrdU incorporation at 36 h after partial hepatectomy), moderately reduced by pioglitazone (15±6%), and not at all suppressed by troglitazone (33±15%), as compared to vehicle control (30±6%, Fig. 1 A, B, *P <0.02 for rosiglitazone vs. vehicle). Consistent with our previously published data (9) , vehicle treatment results in a modest reduction of ~30% vs. no treatment in hepatocellular proliferation at 36 h after partial hepatectomy; however, this difference did not reach statistical significance (data not shown, P =0.1). More extensive analyses of hepatocellular proliferation over times from 24 to 48 h after partial hepatectomy also show greater suppression of hepatocellular proliferation by rosiglitazone than by pioglitazone (Figure 1C , *P <0.04 for vehicle vs. pioglitazone or rosiglitazone; **P <0.02 for vehicle vs. rosiglitazone). However, by 72 h after partial hepatectomy, hepatocellular proliferation in rosiglitazone-treated animals is comparable to that seen in control animals. Taken together, these data demonstrate that thiazolidinediones impair hepatocellular proliferation following partial hepatectomy.


Figure 1
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  		Figure 1. The effects of thiazolidinediones on hepatocellular proliferation following partial hepatectomy. A) Hepatocellular BrdU immunohistochemistry and histology (hematoxylin and eosin staining, H&E) of liver harvested 36 h after partial hepatectomy from vehicle- (Veh), troglitazone- (Tro), pioglitazone- (Pio), and rosiglitazone- (Rosi) treated mice (50-µm scale bar is shown in the upper left panel). B) Summary of hepatocellular proliferation 36 h after partial hepatectomy (*P<0.02 for vehicle vs. rosiglitazone). C) Summary of hepatocellular proliferation at serial times after partial hepatectomy. (*P<0.04 for vehicle vs. pioglitazone or rosiglitazone; **P<0.02 for vehicle vs. rosiglitazone)

To provide further evidence for the suppressive action of thiazolidinediones on liver regeneration, the effects of these drugs on hepatic cyclin expression and mitotic progression following partial hepatectomy were determined. Cyclin D1 regulates G1-S phase cell cycle progression, and its expression is induced by 24 h after partial hepatectomy during normal liver regeneration (36) . Rosiglitazone treatment significantly reduces the expression of cyclin D1 mRNA (Fig. 2 A, *P <0.04 vs. vehicle) and protein (Fig. 2B, C , **P <0.01 vs. vehicle) at this time point. Pioglitazone results in intermediate suppression of both gene and protein expression (Fig. 2A , P =0.06; Fig. 2B, C , *P <0.02), while troglitazone does not suppress the expression of either (Fig. 2A , P =0.4;Fig. 2B, C , P =0.7). These data are consistent with the relative effects of troglitazone, pioglitazone, and rosiglitazone on hepatocellular proliferation (Fig. 1A, B ). Cyclin B1 regulates G2-M phase cell cycle progression and its expression is induced by 48 h after partial hepatectomy (33) . Both pioglitazone and rosiglitazone administration resulted in reduced expression of cyclin B1 mRNA and protein at this time point; however, only the rosiglitazone effect on cyclin B1 protein expression reached statistical significance (Fig. 2D-F , *P<0.04). Similar to its effect on hepatocellular proliferation, vehicle treatment resulted in an ~30% reduction compared to no treatment in cyclin D1 and cyclin B1 protein levels, with neither of these differences reaching statistical significance (P =0.5 for cyclin D1, P =0.1 for cyclin B1, data not shown). Finally, analysis of mitotic body frequency at 48 h after partial hepatectomy, which is the time point corresponding to peak mitotic frequency in untreated C57BL/6J mice (31) , showed greater reduction by rosiglitazone (2±1 mitoses per high powered field) than by pioglitazone (25±14) compared to vehicle control (40±4). Taken together, these observations indicate that thiazolidinediones impair hepatocellular BrdU incorporation, cyclin expression, and mitotic progression during liver regeneration with efficacies that correspond to their potencies of PPAR{gamma}-activation (22 , 26) .


Figure 2
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  		Figure 2. The effects of thiazolidinediones on cyclin expression following partial hepatectomy. A) Hepatic cyclin D1 mRNA expression 24 h after partial hepatectomy in vehicle- (Veh), troglitazone (Tro), pioglitazone- (Pio), and rosiglitazone- (Rosi) treated mice (relative to expression in unoperated livers from vehicle-treated mice; *P <0.04 vs. vehicle); immunoblot (B) and densitometric analysis (C) of hepatic cyclin D1 protein expression 24 h after partial hepatectomy (*P <0.02 and **P <0.01 vs. vehicle). D) Hepatic cyclin B1 mRNA expression is shown 48 h after partial hepatectomy (relative to expression in unoperated liver from vehicle-treated mice), and immunoblot (E) and densitometric analysis (F) of hepatic cyclin B1 protein expression 48 h after partial hepatectomy (*P<0.04 vs. vehicle).

Thiazolidinediones administered at doses that inhibit liver regeneration do not cause hepatotoxicity
In order to establish the specificity of the activity of thiazolidinediones on hepatic regeneration, the effects of these drugs on liver histology, plasma aminotransferase levels, hepatocellular BrdU incorporation, and animal survival were assessed in hepatectomized and unoperated animals subjected to identical dosing regimens as those used for the experiments described above. The results showed that following 5 days of vehicle or drug administration (corresponding to the 72 h posthepatectomy time point), neither hepatectomized (Fig. 1A ) nor unoperated (Fig. 3 A, top) animals exhibit histological evidence of drug-induced hepatic tissue injury or plasma aminotransferase elevation (Fig. 3B ). In addition, unoperated animals subjected to this dosing regimen do not exhibit increased hepatocellular BrdU incorporation, which is the hallmark hepatic response to injury (Fig. 3A , bottom). Finally, thiazolidinedione administration does not result in increased mortality in either unoperated (no mortality) or hepatectomized (vehicle 2% mortality, troglitazone 0%, pioglitazone 8%, rosiglitazone 5%, P =0.5) animals. Together, these data indicate that the suppressive effects of thiazolidinediones on liver regeneration are unlikely to be secondary to drug-induced hepatotoxicity.


Figure 3
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  		Figure 3. The effects of thiazolidinediones on liver histology and plasma aminotransferase activity. A) Hepatocellular BrdU immunohistochemistry and histology (H&E) of liver from unoperated mice treated with vehicle, pioglitazone, or rosiglitazone for 5 days. This corresponds to the 72 h time point for the data shown in Fig. 1 . (A 50-µm scale bar is shown in the left panel). B) Mean and range of plasma alanine aminotransferase (SGPT) determinations on duplicate vehicle- (Veh), pioglitazone- (Pio), and rosiglitazone- (Rosi) treated- hepatectomized and unoperated mice after 5 days of drug administration, which corresponds to the 72 h posthepatectomy time point.

Rosiglitazone does not effect early TNF{alpha}-IL6-STAT3 signaling during liver regeneration
To begin to identify the mechanistic basis for the inhibitory activity of thiazolidinediones on liver regeneration, the effects of the most potent of these drugs, rosiglitazone, on signaling events known to be important for normal liver regeneration were investigated. Activation of the TNF{alpha}-IL6-STAT3 signaling pathway was examined first. This analysis showed that plasma levels of TNF{alpha} and IL6 levels are comparably induced over the initial 6 h following partial hepatectomy in rosiglitazone-treated and control mice (Fig. 4 A, B), indicating that the inhibitory effect of this drug on liver regeneration does not result from suppression of activation of this cytokine signaling cascade. Moreover, by 12 h after surgery, levels of TNF{alpha} and IL6 are significantly greater in rosiglitazone- compared to vehicle-treated animals (Fig. 4A-C ; *P <0.05, **P <0.02). This effect is specific for animals subjected to partial hepatectomy, as rosiglitazone administration does not result in augmented plasma levels of TNF{alpha} or IL6 in sham-operated animals (Fig. 4C ). Similarly, comparable induction of phosphorylated STAT3, a downstream target of IL6, was observed in rosiglitazone- compared to vehicle-treated mouse liver over the first 6 h after partial hepatectomy, with significantly increased levels of activated STAT3 seen in rosiglitazone-treated mice 12–24 h after surgery (Fig. 5 A, B, *P <0.05). Again, this effect was specific for animals subjected to partial hepatectomy, as rosiglitazone did not augment hepatic STAT3 activation in sham operated animals (Fig. 5C , and data not shown).


Figure 4
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  		Figure 4. The effects of rosiglitazone on TNF{alpha} and IL6 induction following partial hepatectomy. Summary of determination of plasma TNF{alpha} (A) and IL6 levels (B) in vehicle- (Veh) and rosiglitazone- (Rosi) treated mice at serial times after partial hepatectomy. C) Plasma TNF{alpha} and IL6 levels in vehicle-, pioglitazone-, or rosiglitazone-treated mice 12 h after partial hepatectomy (Veh, Pio, Rosi) or sham (Veh sham, Rosi sham) surgery. (*P<0.05, **P<0.02, ***P <0.005)


Figure 5
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  		Figure 5. The effects of rosiglitazone on STAT3 activation following partial hepatectomy. A) Protein immunoblot and densitometric analysis (B) of hepatic STAT3 activation at serial times after partial hepatectomy in vehicle (Veh) and rosiglitazone- (Rosi) treated mice (*P <0.05). C) Densitometric analysis of hepatic STAT3 activation in vehicle-, pioglitazone-, or rosiglitazone-treated mice 12 and 24 h after partial hepatectomy (Veh, Pio, Rosi) or sham (Veh sham, Rosi sham) surgery. D) Hepatic SOCS3 mRNA expression after partial hepatectomy in vehicle- (Veh) and rosiglitazone- (Rosi) treated mice.

To determine whether the relative potencies of thiazolidinediones on hepatocellular BrdU incorporation and cyclin expression correspond with their late effects on cytokine signaling, the TNF{alpha}-IL6-STAT3 signaling cascade was also examined in pioglitazone-treated mice 12–24 h after hepatectomy. The results showed that pioglitazone administration results in increased circulating TNF{alpha} levels comparable to those seen with rosiglitazone treatment 12 h after partial hepatectomy (Fig. 4C , ***P <0.005 vs. vehicle, P =0.2 vs. rosiglitazone). Pioglitazone induces more modest changes than rosiglitazone on IL6 induction 12 h after hepatectomy (Fig. 4C , P =0.2 vs. vehicle, P =0.3 vs. rosiglitazone) and on STAT3 activation 12–24 h after hepatectomy (Fig. 5C , P >0.3 vs. vehicle). These data suggest that thiazolidinediones augment TNF{alpha}-IL6-STAT3 signaling 12–24 h after partial hepatectomy with efficacies corresponding to their potencies of PPAR{gamma} activation. On the basis of these observations, the effects of rosiglitazone on expression of suppressor-of-cytokine-signaling (SOCS)-3 mRNA, which is a negative regulator of activated STAT3 induced during liver regeneration (37) , was determined. The result showed that SOCS3 gene expression in regenerating liver is not suppressed by rosiglitazone administration (Fig. 5D ), indicating that rosiglitazone augmentation of hepatic STAT3 activation does not result from impaired SOCS3 induction.

Rosiglitazone inhibits early p38 MAP kinase activation during liver regeneration
Next, the effects of rosiglitazone on p42/44 and p38 MAP kinase activation during liver regeneration were determined. The activities of these MAP kinases are known to be regulated during and thought to be essential for normal liver regeneration (10 , 31) . Moreover, thiazolidinediones have been reported to suppress TNF{alpha}-dependent activation of these MAP kinases in cell culture models (38) . This analysis showed a reduction in the relative activation (phosphorylation) of both the p42/44 and p38 MAP kinases 2–4 h after partial hepatectomy in rosiglitazone- compared to vehicle-treated mice; however, only in the case of p38 did this difference reach statistical significance (Fig. 6 A–D, **P <0.02). Neither the subsequent dephosphorylation of hepatic p38 (6 h after partial hepatectomy) nor the later reactivation (24–36 h after hepatic resection) is effected by rosiglitazone administration (Fig. 6C, D , (31) ). As was also seen with TNF{alpha}, IL6, and STAT3 signaling, rosiglitazone augments the level of phosphorylated p42/44 24 h after partial hepatectomy (Fig. 6A, B , *P <0.002) but has no such effect after sham surgery (data not shown).


Figure 6
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  		Figure 6. The effects of rosiglitazone on p42/44 and p38 MAP kinase activation following partial hepatectomy. Protein immunoblot and densitometric analyses of hepatic p42/44 (A, B) and p38 (C, D) MAP kinase activation at serial times after partial hepatectomy in vehicle- (Veh) and rosiglitazone- (Rosi) treated mice. (*P <0.002, **P <0.02)

Rosiglitazone does not effect C/EBP expression during liver regeneration
The expression patterns of the CCAAT/Enhancer binding Proteins (C/EBPs) {alpha}, ß, and {delta} are known to be regulated during the hepatic regenerative response, with C/EBP{alpha} levels declining and C/EBPß and C/EBP{delta} levels increasing over the initial 24 h following partial hepatectomy. Although still debated, several observations have suggested that such regulation may be important for liver regeneration to proceed normally (33 , 39) . Furthermore, C/EBPß has been shown to regulate PPAR{gamma} expression which, in turn, regulates C/EBP{alpha} expression in models of adipocyte differentiation (40 41 42) . Therefore, in order to further characterize the molecular events associated with rosiglitazone-mediated inhibition of hepatic regeneration, the effects of this drug on mRNA expression of these CCAAT enhancer binding proteins (C/EBP) during liver regeneration were investigated. The results of this analysis showed that rosiglitazone has no significant effect on hepatic C/EBP{alpha} or C/EBPß expression over the initial 24 h following partial hepatectomy. C/EBP{delta} expression is increased in rosiglitazone-treated liver 24 h after hepatectomy but is not affected by drug treatment prior to that (Fig. 7 , *P <0.01). Taken together, these data show that the inhibitory effect of rosiglitazone on hepatic regeneration is not mediated by preventing either induction of C/EBPß and C/EBP{delta} expression or suppression of C/EBP{alpha} expression during early liver regeneration.


Figure 7
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  		Figure 7. The effects of rosiglitazone on C/EBP mRNA expression following partial hepatectomy. Hepatic C/EBP{alpha} (top), C/EBPß (middle), and C/EBP{delta} (bottom) mRNA expression at serial times after partial hepatectomy in vehicle- (Veh) and rosiglitazone- (Rosi) treated mice (*P <0.01).

Rosiglitazone suppresses Skp2 mRNA expression but does not effect p21Waf1/Cip1 or p27Kip1 protein levels during liver regeneration
Troglitazone has been reported to suppress cellular proliferation in culture via a mechanism involving inhibition of mRNA expression of the SCF ubiquitin-ligase complex Skp2 and subsequent accumulation of specific cell-cycle inhibitory targets of Skp2-dependent degradation, including p21Waf1/Cip1 and p27Kip1 (43 44 45 46) . Therefore, the effects of thiazolidinedione administration on Skp2 gene expression and p21Waf1/Cip1 and p27Kip1 protein accumulation during liver regeneration were investigated. Once again, the effects of rosiglitazone were investigated first. This analysis showed that hepatic Skp2 mRNA expression, which is maximally induced between 24 and 48 h after partial hepatectomy, is markedly suppressed by rosiglitazone administration (Fig. 8 A, *P <0.03); however, under these conditions and during this timeframe steady state protein levels of p21Waf1/Cip1 (Fig. 8B, C ) and p27Kip1 (Fig. 8B, D ) are not augmented. The level of p21Waf1/Cip1 protein is increased in rosiglitazone-treated mice at 72 h after surgery (Fig. 8C , *P <0.01); however, this effect is too late to account for the earlier effects of rosiglitazone on hepatocellular proliferation. The effects of troglitazone and pioglitazone on these events were also investigated at the 36 h time point, corresponding to peak Skp2 mRNA induction. The results showed that neither of these drugs has any significant effect on either Skp2 mRNA expression or p21 or p27 protein expression at this time point (data not shown). Together, these data indicate that the effects of rosiglitazone on hepatocellular proliferation during liver regeneration do not result from impaired degradation of the cell cycle inhibitors p21Waf1/Cip1 and p27Kip1, but leave open the possibility that thiazolidinedione effects are mediated by suppression of some other Skp2-dependent activity, such as targeted degradation of other cell-cycle regulators.


Figure 8
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  		Figure 8. The effects of rosiglitazone on Skp2 mRNA and p21Waf1/Cip1 and p27Kip1 protein expression following partial hepatectomy. A) Hepatic Skp2 mRNA expression at serial times after partial hepatectomy in vehicle- (Veh) and rosiglitazone- (Rosi) treated mice (*P <0.03). B) Immunoblot and densitometric (C) analysis of p21Waf1/Cip1 and p27Kip1 protein accumulation at serial times after partial hepatectomy (*P <0.01).

Discussion
The studies reported here show that the thiazolidinedione rosiglitazone impairs liver regeneration. This drug is known to elicit many of its effects through binding and activation of the nuclear steroid hormone receptor PPAR{gamma}. Although PPAR{gamma}-independent effects of the thiazolidinediones have also been described (47) , the observations reported here showing that the relative inhibitory activities of rosiglitazone, pioglitazone, and troglitazone on hepatocellular proliferation and cyclin expression following partial hepatectomy correspond precisely with the relative PPAR{gamma}-activating potencies of these drugs (22 , 25 26 27) support the conclusion that the inhibitory effect of rosiglitazone on liver regeneration is mediated through its interaction with PPAR{gamma}. Thus, these data implicate, for the first time, PPAR{gamma} as an important candidate regulator of the hepatic regenerative response.

The data reported here do not distinguish between the possibilities that the inhibitory effects of rosiglitazone on hepatic regeneration depend on drug-interactions with hepatic vs. extrahepatic PPAR{gamma}. Indeed, under normal physiological conditions, levels of hepatic PPAR{gamma} expression appear to be low, and the functional importance of such expression has not been clearly elucidated (24 , 48) . However, the observation that hepatic PPAR{gamma} expression is increased in genetically and environmentally induced models of fatty liver disease (49 50 51) suggests that hepatic PPAR{gamma} expression can be an important determinant of liver-specific physiology and pathophysiology.

Our data indicate that the mechanisms responsible for the inhibitory effect of rosiglitazone on liver regeneration do not depend on suppression of early cytokine signaling. Indeed, plasma levels of TNF{alpha} and IL6 and hepatic levels of activated STAT3 were unchanged over the first 6 h after hepatectomy and were augmented 12–24 h after hepatectomy in rosiglitazone- vs. vehicle-treated animals. These data would seem to conflict with published observations showing that thiazolidinediones can suppress lipopolysaccharide (LPS)-stimulated TNF{alpha} production (52 53 54) ; however, this apparent contradiction can be reconciled by a recent report indicating that proinflammatory cytokine production during liver regeneration is independent of LPS signaling (55) . Furthermore, our data, taken together with published data indicating that IL6 supplementation can suppress hepatocellular proliferation in wild-type (WT) mice subjected to partial hepatectomy (6) , raise the possibility that the impaired regenerative response in rosiglitazone-treated animals may even be the result of increased cytokine signaling. Rosiglitazone does suppress the early activation but not later changes in the activity of hepatic p38 MAP kinase during liver regeneration; however, the functional significance of regulated p38 MAP kinase activity during the regenerative response has not yet been elucidated. The data presented here also indicate that the impaired regenerative response seen in rosiglitazone-treated mice does not result from disruption of regulation of C/EBP gene expression, or impaired degradation of the cell cycle inhibitors p21Waf1/Cip1 or p27Kip1. Our data showing that thiazolidinediones inhibit cyclin D1 expression (Figure 2A-C ), raise the possibility, as suggested by analyses in cell culture (56 57 58 59) , that direct PPAR{gamma}-dependent inhibition of cyclin expression may be responsible for the effects of rosiglitazone on liver regeneration.

Finally, our data have important potential implications with respect to the hepatotoxicity associated with clinical use of thiazolidinediones. Thiazolidinediones were developed as antidiabetic, insulin-sensitizing drugs (22, 26). Troglitazone, which was the first of these drugs used to treat patients with insulin-resistant diabetes mellitus, was associated with the development of idiosyncratic acute liver failure, and therefore withdrawn from clinical use (60, 61). Hepatotoxicity has subsequently been reported in patients taking pioglitazone and rosiglitazone, raising the possibility that thiazolidinediones exert class-specific hepatotoxicity through their effects on PPAR{gamma} activity (61, 62). Although the mechanistic basis for such toxicity remains entirely unknown, the studies described here raise the possibility that thiazolidinedione-mediated hepatotoxicity results from PPAR{gamma}-dependent inhibition of hepatic regeneration, and provide support for current clinical practices in which these drugs are avoided or used judiciously in patients with known or suspected liver disease.

ACKNOWLEDGMENTS

The authors thank Drs. Lou Muglia, Jonathan Gitlin, and Alex Weymann for helpful suggestions and critical review of this manuscript. These studies were supported by grants to DAR from NIH (DK02900, DK068219) and March of Dimes (Basil O’Connor Award), and by the Digestive Disease Research Core Center (NIH grant # P30 DK52574). YPT was supported in part by Institutional Training Grant T32-HD07409.

Received for publication May 15, 2006. Accepted for publication August 7, 2006.

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