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Mice subjected to partial hepatectomy (PH) develop hypoglycemia, followed by increased systemic lipolysis and hepatic fat accumulation, prior to onset of hepatocellular proliferation. Strategies that disrupt these metabolic events inhibit regeneration. These observations suggest that alterations in metabolism in response to hepatic insufficiency promote liver regeneration. Hepatic expression of the peroxisome proliferator-activated receptor gamma (PPARγ) influences fat accumulation in the liver. Therefore, the studies reported here were undertaken to assess the effects of disruption of hepatic PPARγ expression on hepatic fat accumulation and hepatocellular proliferation during liver regeneration. The results showed that liver regeneration was not suppressed, but rather modestly augmented in liver-specific PPARγ null mice maintained on a normal diet. These animals also exhibited accelerated hepatic cyclin D1 expression. Because hepatic PPARγ expression is increased in experimental models of fatty liver disease in which liver regeneration is impaired, regeneration in liver-specific PPARγ null mice with chronic hepatic steatosis was also examined. In contrast to the results described above, disruption of hepatic PPARγ expression in mice with diet-induced hepatic steatosis resulted in significant suppression of hepatic regeneration. Conclusion: The metabolic and hepatocellular proliferative responses to PH are modestly augmented in liver-specific PPARγ null mice, thus providing additional support for a metabolic model of liver regeneration. Furthermore, regeneration is significantly impaired in liver-specific PPARγ null mice in the setting of diet-induced chronic steatosis, suggesting that pharmacological strategies to augment hepatic PPARγ activity might improve regeneration of the fatty liver. (HEPATOLOGY 2012)
The liver has a remarkable capacity to regenerate. Studies using the partial hepatectomy (PH) model have shown that this response to hepatic insufficiency is precisely regulated, and results in restoration of the original liver/body mass ratio. Experimental analyses in pharmacologically and genetically manipulated mice using PH and other models have identified many signals essential for normal liver regeneration. These signals include cytokines, growth factors, intracellular signaling events, and transcription factors, which direct the induction of specific gene expression programs that induce hepatocellular proliferation and restore liver mass (reviewed in Michalopoulos,1, 2 Michalopoulos and DeFrances,3 Diehl and Rai,4 and Fausto5). Despite such knowledge, the signals that initiate and terminate hepatic regeneration are still incompletely defined.
Several observations implicate the metabolic response to hepatic insufficiency in the regulation of liver regeneration.6 Mice subjected to PH rapidly develop hypoglycemia,7 followed by increased systemic catabolism, elevated serum free fatty acids (FFAs),8 and hepatic accumulation of lipids9, 10 and amino acid catabolites.11 Experimental strategies that disrupt these events, such as dextrose supplementation or suppression of hepatic fat accumulation, inhibit regeneration.7, 9, 12, 13 Furthermore, fatty liver dystrophy (fld) mice, which exhibit significantly reduced adipose stores,14 develop attenuated hypoglycemia, reduced hepatic fat accumulation, and impaired regeneration after PH.10 Taken together, these observations support a model in which the hypoglycemic response to PH causes adipose tissue lipolysis followed by hepatic accumulation of the resulting metabolites, and that these events induce quiescent hepatocytes to proliferate until the normal liver/body mass ratio is restored.15 Moreover, comparable alterations in hepatic and systemic metabolism have been reported in toxin-induced models of liver regeneration,6 raising the intriguing hypothesis that the metabolic response to hepatic insufficiency is the proximal event that promotes liver regeneration after any form of hepatic injury.
The peroxisome proliferator-activated receptor gamma (PPARγ) is an important determinant of hepatic fat accumulation, as demonstrated by analyses showing that hepatic PPARγ expression is augmented in experimental models of fatty liver disease,16-19 and that genetic disruption of such expression leads to diminished hepatic steatosis in some of these models.20, 21 Based on those observations and the considerations discussed above, the studies reported here were undertaken to assess the effects of liver-specific disruption of PPARγ expression on hepatic fat accumulation and hepatocellular proliferation during normal liver regeneration and on regeneration in the setting of diet-induced fatty liver disease using the PH model.
Pparγ-loxP mice (The Jackson Laboratory, Bar Harbor, ME)22 were bred to mice engineered for hepatocyte-specific Cre recombinase expression regulated by the albumin promoter [Alb-Cre; strain B6.Cg-Tg(Alb-Cre)21Mgn/J, The Jackson Laboratory] to generate mutant Pparγfl/fl-Alb-Cre+ mice and Pparγfl/fl-Alb-Cre− controls. Liver-specific disruption of PPARγ expression was confirmed as described in the Supporting Information. At weaning, ad libitum access to either a standard diet (Mouse Diet 20; LabDiet PicoLab, Brentwood, MO) or steatogenic diet (TD.88137 “Western” Adjusted Calories Diet; Harlan Teklad, Madison, WI) was provided. Some animals were given supplemental dextrose as described.7 At 8-12 weeks of age, mice were subjected to two-thirds partial hepatectomy (PH) or sham surgery as described in the Supporting Information and elsewhere.7, 9, 10, 24-27 At serial times after surgery, animals were sacrificed, and plasma and liver tissue were harvested. Three or more animals were examined at each time point for each genotype, surgical, and treatment group. 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” (National Institutes of Health publication 86-23).
Liver Histology, Immunohistochemistry, Messenger RNA, and Protein Expression, Triglyceride Content, and Serum FFAs and Glycerol Analyses.
These analyses were conducted as described in the Supporting Information and elsewhere.7, 9, 10, 24-28
Numerical data comparisons between groups were conducted using an unpaired Student t test or Mann-Whitney rank sum test for pairwise comparisons, and analysis of variance (ANOVA) or ANOVA on ranks for multiple groups. Rates and proportions were compared using a chi-square test. Significance (alpha) was set at 0.05. Data are reported as the mean ± SE.
The Regenerative Response to PH Is Accelerated in Liver-Specific PPARγ Null Mice.
The regulation and functional importance of hepatic PPARγ expression during normal liver regeneration was determined by comparing the response to PH between liver-specific Pparγ null mice (Pparγfl/fl-Alb-Cre+) and their Cre− littermate controls. Liver-specific disruption of PPARγ expression was confirmed using both a polymerase chain reaction (PCR) assay that distinguishes expression of the wild-type and (Cre-dependent) truncated mutant forms of PPARγ1 (Fig. 1A)22 and a semiquantitative real-time reverse-transcription PCR (qRT-PCR)–based assay for expression of total PPARγ, PPARγ1, and PPARγ2 messenger RNA (mRNA) expression (Fig. 1B). mRNA expression of the mutant allele was detectable in liver tissue from Cre+ mice, whereas the wild-type allele was present in the livers of Cre− mice and in extrahepatic tissues of Cre+ mice (Fig. 1A). qRT-PCR analysis showed suppression of 90% of total PPARγ, PPARγ1, and PPARγ2 expression in quiescent liver tissue from Cre+ mice compared with that in Cre− controls (Fig. 1B). The regulation of hepatic PPARγ mRNA expression in response to PH was also investigated. The results showed an early rise and a subsequent decline in total PPARγ and PPARγ1 expression, and an early decrease with subsequent increase in PPARγ2 expression in regenerating livers from Cre− mice (Fig. 1C-E). As expected, the expression of each isoform was significantly reduced in regenerating livers from Cre+ animals (Fig. 1C-E); however, a small amount of residual expression was present in quiescent and regenerating liver (Fig. 1B-E), likely from nonparenchymal liver cells (e.g., stellate, endothelial, and Kupffer cells) or incomplete hepatocellular Cre-mediated deletion.
Analysis of liver regeneration showed that the hepatocellular proliferative response to PH was not impaired in Cre+ mice (Fig. 2A). In fact, regeneration was modestly but significantly augmented in these animals, with increased hepatocellular bromodeoxyuridine (BrdU) incorporation 36 hours after surgery compared with Cre− littermate controls (Fig. 2A; 32% ± 2% versus 22% ± 4%). This timepoint corresponds to peak proliferation during liver regeneration in wild-type mice (Fig. 2A).7, 10, 26-28 Hepatocellular mitotic frequency, which is not detectable 36 hours and peaks 48 hours after PH (Fig. 2B),7, 27 and recovery of liver/body mass ratio (Supporting Fig. 1) after PH were comparable between Cre+ and Cre− mice. Postoperative mortality occurred in 0/24 Cre+ mice and 4/25 Cre− mice (P = 0.04). Together, these data demonstrate that liver regeneration is not compromised in liver-specific PPARγ null mice, and even appears to be modestly accelerated.
Disruption of Hepatic PPARγ Expression Does Not Prevent Hypoglycemia, Weight Loss, Serum FFA Elevation, or Hepatic Triglyceride Accumulation in Response to PH.
Next, alterations in metabolism that occur in response to PH-induced hepatic insufficiency during normal regeneration6, 7, 9, 10 were examined in liver-specific PPARγ null mice. Body mass (Supporting Fig. 2), blood glucose, and hepatic triglyceride content (Fig. 3A-B, 0 hours) were comparable in Cre+ and Cre− mice at baseline. Following PH, both control and liver-specific PPARγ null mice developed hypoglycemia (Fig. 3A). Both Cre+ and Cre− mice also showed decline in total body mass and accumulation of hepatic triglyceride after PH, with blunted weight loss (Fig. 3B) and a trend toward augmented fat accumulation (Fig. 3C) in the Cre+ (versus Cre−) animals. Serum FFAs increased after PH in wild-type mice (Fig. 3D),8 consistent with PH-induced adipose tissue lipolysis. Thus, the finding that serum FFAs are comparable in regenerating Cre+ and Cre− livers (Fig. 3D), together with the data above, indicate that the metabolic response to PH is not suppressed in Cre+ mice. To further evaluate this conclusion, glycerol, which is also released during adipose lipolysis, was quantified in serum. The results did not demonstrate PH-induced elevation in serum glycerol (data not shown), perhaps because glycerol may be taken up more rapidly than FFAs by regenerating liver. The observation that the PH-induced elevation in serum FFAs is not prevented by disruption of hepatocellular PPARγ expression does not contradict the recognized role of PPARγ expression in adipose lipolysis29 because adipose tissue expression of PPARγ is not disrupted in the Cre+ animals (Fig. 1).
Analyses of Cell Cycle Regulation During Liver Regeneration in Liver-Specific PPARγ Null Mice.
The influence of hepatic PPARγ expression on signals that promote cell cycle progression during regeneration was examined next. Consistent with the analyses of hepatocellular BrdU incorporation and mitosis described above, induction of cyclin D1 mRNA expression, which promotes G1-S phase cell cycle progression, was accelerated in liver-specific null mice (Fig. 4A), but that of cyclin B1, which promotes G2-M phase cell cycle progression, was comparable between Cre+ and Cre− animals (Fig. 4B). Hepatic expression of the forkhead box transcription factor FoxM1, which is induced during and required for normal liver regeneration,30 was examined next. FoxM1 expression is suppressed by PPARγ-activating thiazolidinediones (TZDs) in cell culture.31 Nevertheless, hepatic FoxM1 mRNA expression was comparably induced after PH in Cre+ and Cre− mice (Fig. 4C). TZDs also inhibit the expression of Skp2 mRNA, which encodes a ubiquitin ligase that promotes degradation of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1,32-35 and Skp2 expression is induced during normal liver regeneration.26 Analyses of the patterns of expression of Skp2 mRNA and p21 and p27 proteins in regenerating liver showed no significant differences between liver-specific PPARγ null mice and Cre− controls in the expression of any of these factors (Figs. 4D and 5A,B). These data are consistent with the observation that hepatocellular progression through S phase but not subsequent stages of the cell cycle is augmented in the liver-specific null mice.
Analyses of Adipogenic Gene Expression During Liver Regeneration in Liver-Specific PPARγ Null Mice.
Previous work has shown that hepatic PPARγ overexpression induces adipogenic and lipogenic gene expression in mouse liver.36 Furthermore, some of the gene expression upregulated in that model is also increased in early regenerating liver.9 Therefore, the influence of hepatic PPARγ disruption on adipogenic gene expression in early regenerating liver was investigated. The results showed that adipocyte fatty acid binding protein (aP2) mRNA was induced 24 hours after PH in Cre− but not Cre+ mice (Fig. 6A), and fatty acid synthase (FAS) expression was suppressed in liver-specific PPARγ null mice but was unchanged in controls during early regeneration (Fig. 6B). In contrast, the adipose differentiation-related protein (ADRP or perilipin2), fatty acid translocase (CD36), and FSP-27 (CIDE-C) were each induced (Fig. 6D-F) and fatty acid transport protein 2 (FATP2) was unchanged (Fig. 6C) in both Cre+ and Cre− animals (although basal ADRP expression is higher in tissue-specific null mice, Fig. 6D). These data demonstrate target-specific PPARγ effects on adipogenic gene expression in quiescent and regenerating liver.
Dextrose Supplementation Suppresses Liver Regeneration in Liver-Specific PPARγ Null Mice.
The functional importance of PPARγ expression in systemic glycemic regulation37 raises the possibility that hepatic PPARγ expression modulates the inhibitory effect of dextrose supplementation on liver regeneration.7 To address this question, liver regeneration in Cre+ mice provided with supplemental dextrose was compared with that in unsupplemented Cre+ mice. The results showed that hepatocellular proliferation after PH was comparably reduced by provision of supplemental dextrose in these animals (Supporting Fig. 3) as was reported in wild-type mice.7 Thus, PPARγ expression does not mediate the inhibitory effect of dextrose supplementation on liver regeneration.
Liver Regeneration Is Impaired in Liver-Specific PPARγ Null Mice with Diet-Induced Hepatic Steatosis.
Hepatic PPARγ expression is increased in animal models of hepatic steatosis, including ob/ob, db/db, and KK-Ay mice and mice with diet-induced fatty liver.16-19 Furthermore, liver regeneration is impaired in these models.38-44 Together with the data reported above showing modestly augmented liver regeneration in liver-specific PPARγ null mice, those observations suggest that increased hepatic PPARγ expression could mediate the inhibitory effect of steatosis on regeneration. To address this question, the effect of diet-induced hepatic steatosis on liver regeneration in liver-specific PPARγ null mice was investigated. In these studies, liver-specific null and Cre− control mice were maintained on a steatogenic diet from the time of weaning until age 3 months, at which time they were subjected to PH and analyses of liver regeneration. As expected, triglyceride content (Fig. 7A) and total PPARγ expression (Fig. 7B) were each significantly increased in unoperated livers from control (Cre−) mice maintained on the steatogenic diet compared with those from Cre− mice on a normal diet. The increased total PPARγ expression was largely the result of dietary effects on PPARγ2 expression (Fig. 7B), with PPARγ1 expression not significantly different between mice fed the standard and steatogenic diets. Interestingly, basal hepatic triglyceride content was comparably elevated in Cre+ and Cre− mice by the steatogenic diet (Fig. 7A). Furthermore, neither genotype accumulated additional hepatic fat following PH (Fig. 7A). This observation suggests that the systemic catabolic response to PH, which contributes to fat accumulation in the regenerating liver,10 is at least partially suppressed by effects of the steatogenic diet. Indeed, both Cre+ and Cre− mice maintained on the steatogenic diet exhibited reduced weight loss after PH compared with that seen in mice fed the standard diet (Supporting Fig. 4). Surprisingly, the regenerative response to PH was not significantly impaired by diet-induced hepatic steatosis in the Cre− mice compared with those mice maintained on the control diet in this experiment, with hepatocellular BrdU incorporation 36 hours after PH of 22% ± 4% in Cre− mice on the standard diet versus 20% ± 3% on the steatogenic diet (Fig. 7C,D). Furthermore, examination of the Cre+ animals showed that liver regeneration was not augmented (as on standard chow, Fig. 2A), but rather was significantly impaired in the liver-specific PPARγ null mice on the steatogenic diet, with hepatocellular BrdU incorporation of 16% ± 3% on the steatogenic diet versus 32% ± 2% on the control diet 36 hours after partial liver resection (Fig. 7C,D). Cyclin D1 and FoxM1 mRNA showed trends toward higher expression in regenerating livers from Cre− compared with Cre+ mice on the steatogenic diet; however, these differences did not achieve statistical significance (Fig. 8A,B). Hepatic expression of Skp2 mRNA (Fig. 8C) and p21 and p27 proteins (data not shown) were similar in control and liver-specific null mice on the steatogenic diet. Examination of adipogenic gene expression showed significantly increased aP2, FATP2, and CD36 expression in regenerating livers from Cre+ (but not Cre−) mice on the steatogenic diet (Fig. 8D-F). These data demonstrate that hepatic PPARγ expression does not mediate impaired regeneration but rather promotes the regenerative response in the setting of diet-induced hepatic steatosis. Furthermore, they show that hepatic PPARγ expression is not essential for the development of diet-induced hepatic steatosis but does affect adipogenic gene expression in this model.
The studies reported here were undertaken to investigate the influence of hepatic PPARγ expression on hepatic fat accumulation and hepatocellular proliferation during the regenerative response to PH in the absence and presence of diet-induced fatty liver disease. This question was provoked by our own data implicating the metabolic response to PH (including transient hepatic fat accumulation) in regulating such regeneration,6, 7, 9, 10 and other studies demonstrating a role for hepatic PPARγ expression in promoting fat accumulation in experimental models of fatty liver disease.20, 21 The results showed that neither hepatic fat accumulation nor the regenerative response to PH required hepatocellular PPARγ expression. In fact, the early proliferative response was modestly accelerated by disruption of such expression, as assessed by hepatocellular BrdU incorporation. Mitotic progression was not detectably augmented in Cre+ versus Cre− mice. Circadian effects on hepatocellular G2-M phase but not G1-S phase progression have been reported45 and might contribute to the differences in these parameters in the current study. Nevertheless, the data reported here are consistent with previous studies showing that PPARγ-activating TZDs inhibit liver regeneration.26, 46 These studies also show that the metabolic response to PH is not suppressed in liver-specific PPARγ null mice. The accelerated induction of cyclin D1 expression after PH in liver-specific PPARγ null mice provides some insight into the molecular machinery that promotes augmented regeneration in these animals. However, Skp2 expression, which encodes an E3 ubiquitin ligase that promotes degradation of cyclin-dependent kinase inhibitors, is induced in regenerating liver26 and is suppressed by TZDs in cultured cells,32-35 was comparably induced in regenerating liver from Cre+ and Cre− animals. Thus, the specific mechanisms linking the metabolic response to hepatic insufficiency and initiation of liver regeneration require further investigation.
Hepatic PPARγ expression is augmented in various models of fatty liver disease16-19 in which liver regeneration has been reported to be impaired.38-44 Thus, the accelerated regenerative response in liver-specific PPARγ null mice observed here raised the possibility that augmented PPARγ expression in fatty liver might mediate the impaired regenerative response observed in those settings. Therefore, we examined liver regeneration in liver-specific PPARγ null and Cre− control mice with diet-induced hepatic steatosis. Hepatic triglyceride content was comparably elevated at baseline in Cre+ and Cre− mice; thus, disruption of hepatic PPARγ expression does not prevent diet-induced steatosis. Surprisingly, we did not observe impaired regeneration in the Cre− control mice exposed to the steatogenic diet compared with animals on the control diet. The reasons for the differences in our results and those of the previous report employing this same steatogenic diet, which showed markedly impaired regeneration in wild-type mice with diet-induced steatosis,43 are uncertain. Of note, we previously reported data implicating technical factors (e.g., choice of anesthesia), as influencing morbidity and mortality in the PH model,27 which might be the case here as well. Nevertheless, we were able to assess the impact of disruption of hepatic PPARγ expression on liver regeneration in the setting of diet-induced steatosis. Remarkably, regeneration was significantly suppressed by fatty liver disease in Cre+ mice. These observations are consistent with a recent report describing rescue of impaired regeneration in diabetic KK-Ay mice by treatment with the TZD pioglitazone.42 Future experimental analyses should examine the effects of TZDs on liver regeneration in various genetic and dietary models of fatty liver disease. Such investigation would further evaluate the possibility, suggested by the data reported here, that pharmacological strategies to augment hepatic PPARγ activity might improve regeneration of the fatty liver. Proof of that hypothesis in preclinical analyses would provide support for clinical trials to test the efficacy of TZDs on prevention of adverse outcomes in patients with hepatic steatosis who undergo major hepatic resection.47
We thank Emily Barr for technical support, the Washington University School of Medicine Digestive Diseases Research Core Centers for histological and immunohistochemical support, and the Washington University School of Medicine Nutrition Obesity Research Center for serum-free fatty acid analyses.