p21 is required for dextrose-mediated inhibition of mouse liver regeneration

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

The inhibitory effect of dextrose supplementation on liver regeneration was first described more than 4 decades ago. Nevertheless, the molecular mechanisms responsible for this observation have not been elucidated. We investigated these mechanisms using the partial hepatectomy model in mice given standard or 10% dextrose (D10)-supplemented drinking water. The results showed that D10-treated mice exhibited significantly reduced hepatic regeneration compared with controls, as assessed by hepatocellular bromodeoxyuridine (BrdU) incorporation and mitotic frequency. D10 supplementation did not suppress activation of hepatocyte growth factor (HGF), induction of transforming growth factor alpha (TGF-α) expression, or tumor necrosis factor alpha–interleukin-6 cytokine signaling, p42/44 extracellular signal-regulated kinase (ERK) activation, immediate early gene expression, or expression of CCAAT/enhancer binding protein beta (C/EBPβ), but did augment expression of the mito-inhibitory factors C/EBPα, p21Waf1/Cip1, and p27Kip1. In addition, forkhead box M1 (FoxM1) expression, which is required for normal liver regeneration, was suppressed by D10 treatment. Finally, D10 did not suppress either FoxM1 expression or hepatocellular proliferation in p21 null mice subjected to partial hepatectomy, establishing the functional significance of these events in mediating the effects of D10 on liver regeneration. Conclusion: These data show that the inhibitory effect of dextrose supplementation on liver regeneration is associated with increased expression of C/EBPα, p21, and p27, and decreased expression of FoxM1, and that D10-mediated inhibition of liver regeneration is abrogated in p21-deficient animals. Our observations are consistent with a model in which hepatic sufficiency is defined by homeostasis between the energy-generating capacity of the liver and the energy demands of the body mass, with liver regeneration initiated when the functional liver mass is no longer sufficient to meet such demand. (HEPATOLOGY 2009.)

The liver has remarkable regenerative potential, which permits recovery from functional deficits occurring after hepatic injury.1–3 Partial hepatectomy in rodents has been the most extensively used experimental model for investigating the molecular, cellular, and physiological mechanisms that control this highly regulated response.4 Analyses using this system have led to the identification of a number of signals that are regulated during and necessary for normal liver regeneration. For example, the early hepatic regenerative response is characterized by initiation of Wnt-dependent,5–7 growth factor–dependent,8–12 and cytokine-dependent13–15 signaling, induction of p42/44 extracellular signal regulated kinase (ERK) activity,16 and activation of transcription factors including beta-catenin, nuclear factor kappaB, and signal transducer and activator of transcription protein 3.17–19 These events direct an immediate early gene expression program20 culminating in hepatocellular re-entry into and progression through the cell cycle. Ultimately, this leads to restoration of normal hepatic mass. Despite this knowledge, an integrated understanding of the precise mechanistic regulation of the hepatic regenerative response remains incomplete. Indeed, the nature and identities of the most proximal and distal signals that initiate and terminate hepatic regeneration are largely unknown.

Liver mass is maintained in health or recovered by regeneration after injury in precise proportion to body mass.21 This well-known observation suggests that the signals that initiate and terminate the hepatic regenerative response might be coupled to systemic demands on hepatic function. Consistent with this idea, previous studies have shown that rodents become hypoglycemic after partial hepatectomy and that either intravenous or enteral dextrose supplementation markedly suppresses the hepatic regenerative response.22–26 Although these observations were first made more than 4 decades ago, neither their functional significance nor their mechanistic basis has yet been elucidated. In this manuscript, we describe our analyses of the molecular mechanisms responsible for dextrose-mediated inhibition of liver regeneration.

Abbreviations

BrdU, bromodeoxyuridine; C/EBP, CCAAT enhancer binding protein; D10, 10% dextrose-supplemented water; ERK, extracellular signal regulated kinase; FoxM1, forkhead box M1; GSK3β, glycogen synthase kinase 3β; HGF, hepatocyte growth factor; mRNA, messenger RNA; TGF-α, transforming growth factor alpha.

Materials and Methods

Animal Husbandry and Surgery.

Male 2-month-old to 3-month-old wild-type C57Bl/6J and Cdkn1a (p21)-null mice (B6;129S2-Cdkn1atm1Tyj/J; Jackson Laboratory, Bar Harbor, ME) were maintained on 12-hour dark-light cycles with ad libitum access to standard rodent chow and water until 60 hours before surgery. At that time, experimental mice were provided ad libitum access to sterile-filtered 10% dextrose (D10) in drinking water, whereas control animals were given unsupplemented sterile water. Access to chow was continued in both groups, and D10 and unsupplemented water were changed daily. Mice were subjected to partial hepatectomy using standard methodology27–30: Mice were sedated with inhaled isoflurane (VEDCO Inc., St. Joseph, MO) via anesthesia vaporizer, then subjected to mid-ventral laparotomy with exposure, ligation, and resection of the left and median hepatic lobes, and closure of the peritoneal and skin wounds. At serial times after surgery, animals were sacrificed by inhaled CO2, and plasma and liver tissue were harvested. Blood glucose was determined by standard glucometric analysis (Ascensia Contour, Bayer Healthcare, Tarrytown, NY) immediately before sacrifice. Five to 15 animals were examined at each time point for each genotype 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” (NIH publication 86-23).

Histology and Immunohistochemistry.

Liver histology and hepatocellular bromodeoxyuridine (BrdU) incorporation were assessed as described in Supporting Materials and previously.27–29

Plasma Cytokine and Insulin Determination.

Tumor necrosis factor alpha and interleukin-6 levels were determined using the Bio-Plex multiplex bead–based assay (Bio-Rad Laboratories, Hercules, CA), and insulin levels were measured using a commercially available enzyme-linked immunoassay (Linco, St. Charles, MO), each according to the manufacturer's instructions.

Protein Expression Analysis.

Liver protein lysates were made and protein immunoblot studies performed and quantified as previously described.27 Primary antibodies are listed in Supporting Materials.

Gene Expression Analysis.

Total RNA was analyzed for expression of specific genes of interest using real-time reverse-transcriptase polymerase chain reaction as described in Supporting Material and previously.28 Target-specific forward and reverse primers are listed in Supporting Table 1.

Statistical Analysis.

Data were analyzed using SigmaPlot and SigmaStat software (SPSS, Chicago, IL). Unpaired Student t test for pair-wise comparisons and analysis of variance for multiple groups were used to compare hepatocellular BrdU incorporation, mitotic body frequency, liver weight, and messenger RNA (mRNA) and protein expression levels between experimental groups, with significance (alpha) set at 0.05. Data are reported as mean ± standard error.

Results

Dextrose Supplementation Inhibits Liver Regeneration.

To investigate the functional significance of the hypoglycemia that occurs after partial hepatectomy and the mechanistic basis for the inhibitory effect of dextrose supplementation on liver regeneration,22–26 we first characterized the kinetics of the murine hypoglycemic response to partial hepatectomy. The results showed that mice developed significant hypoglycemia detectable by 3 hours, persisting through 72 hours, and resolved by 7 days after surgery (Supporting Fig. 1). Next, to characterize the inhibitory effect of dextrose supplementation on liver regeneration, the hepatocellular proliferative response to partial hepatectomy was compared between mice offered 10% dextrose-supplemented water (D10) and those given unsupplemented water. Water intake in D10-treated mice was approximately fourfold higher (17 ± 0.6 mL/animal/day) than in controls (4.7 ± 0.4, *P < 0.001). Analysis of the regenerative response showed significantly reduced hepatocellular BrdU incorporation in D10-treated mice 36 hours after partial hepatectomy (Fig. 1A,B; P < 0.02), which is the timepoint corresponding to peak proliferation during liver regeneration in wild-type mice (Fig. 1B).27 Hepatocellular mitoses were also reduced in these animals at 48 hours after surgery (Fig. 1C,D; *P < 0.04), the time of peak mitotic progression during normal liver regeneration (Fig. 1C,D).29 Liver: body weight 72 hours after surgery was approximately 10% lower in D10-treated animals (3.3% ± 0.3%) compared with controls (3.7 ± 0.1); however, this difference did not reach statistical significance (P = 0.22). The effect of D10 supplementation on hypoglycemia after partial liver resection was also analyzed, with results showing increased plasma glucose in D10-supplemented animals at 36 hours after partial hepatectomy (Fig. 1E; P < 0.003), but not at other time-points from 0 to 72 hours after surgery. Insulin levels were also increased at this timepoint (Fig. 1F); however, this difference was not significant (P = 0.06). These results are consistent with prior analysis of dextrose-mediated inhibition of liver regeneration25 and suggest that dextrose supplementation must influence glucose flux into liver, extrahepatic tissues, or both during the regenerative response.

Figure 1.

Liver regeneration in D10-supplemented mice. (A) Immunohistochemical analysis of hepatocellular BrdU incorporation (upper panels) and histological analysis (hematoxylin-eosin [H&E], lower panels) of liver 36 hours after partial hepatectomy in control (water) and dextrose (D10)-supplemented mice (100-μm bar in upper left panel). (B) Summary of hepatocellular proliferation (fraction of total hepatocytes that incorporated BrdU) 24 to 72 hours after partial hepatectomy in regenerating liver from control and D10-supplemented mice (*P < 0.02). (C) HE staining and (D) summary of mitotic body frequency 48 hours after partial hepatectomy (*P < 0.04). (E) Blood glucose and (F) plasma insulin levels after partial hepatectomy (*P < 0.003).

D10 Supplementation Does Not Inhibit HGF Activation or TGF-α Expression During Liver Regeneration.

To begin to investigate the mechanisms responsible for D10-mediated inhibition of liver regeneration, the influence of dextrose on growth factor–dependent signaling was investigated. This analysis showed that expression and activation of hepatocyte growth factor (HGF), which promotes normal liver regeneration through activation of hepatocyte growth factor receptor (c-Met)8–11 were comparable in control and D10-treated animals subjected to partial hepatectomy (Fig. 2A-C). Furthermore, p42/44 ERK activation, which is dependent on hepatocyte growth factor receptor (c-Met) signaling during liver regeneration,11 was not disrupted by D10 supplementation (Fig. 2D). Finally, expression of the epidermal growth factor receptor ligand transforming growth factor alpha (TGF-α), which increases in liver after partial hepatectomy,31 was comparable in regenerating liver from D10-treated and control animals (Fig. 2E,F). These data show that D10 does not inhibit activation of HGF or expression of TGF-α in regenerating liver.

Figure 2.

Growth factor expression and activation and ERK activation during liver regeneration in D10-supplemented mice. (A) HGF mRNA and (B, C) mature α-HGF protein expression and densitometric analysis after partial hepatectomy (*P < 0.05 versus 0 hours). (D) Representative protein immunoblot for hepatic phosphorylated and total p42/44 ERK after partial hepatectomy. (E) TGF-α mRNA expression and (F) representative TGF-α protein immunoblot after partial hepatectomy (*P < 0.02 versus 0 hours).

D10 Supplementation Does Not Suppress Glycogen Synthase Kinase 3β Activation or β-Catenin Regulated Gene Expression During Liver Regeneration.

Hepatic beta-catenin expression is required for normal liver regeneration6, 7 and can augment epidermal growth factor receptor signaling.32 Canonical activation of this pathway requires Wnt-dependent inhibition (by phosphorylation) of glycogen synthase kinase 3β (GSK3β), which otherwise phosphorylates and promotes the degradation of beta-catenin.33 GSK3β also phosphorylates and inhibits glycogen synthase.34 Thus, GSK3β regulates pathways involved in liver regeneration and glucose homeostasis. Therefore, the effects of D10 supplementation on GSK3β phosphorylation and β-catenin−dependent gene expression were examined. The results showed that D10 did not suppress hepatic GSK3β phosphorylation. In fact, higher levels of phosphorylated GSK3β were seen in livers from D10-treated versus control animals (Fig. 3A,B; *P < 0.01). Similarly, D10 did not impair transcriptional induction of many beta-catenin targets,7, 33 including Axin2 (Fig. 3C), c-Fos, c-Myc (Supporting Fig. 3A,C), and cyclin D (Fig. 5A). Thus, D10 does not disrupt GSK3β regulation of beta-catenin–dependent gene expression during liver regeneration.

Figure 3.

GSK3β activation during liver regeneration in D10-supplemented mice. (A) Protein immunoblot and (B) densitometric analysis of hepatic phosphorylated and total GSK3β after partial hepatectomy (*P < 0.01). (C) Axin2 mRNA expression after partial hepatectomy.

Figure 5.

Cyclin expression during liver regeneration in D10-supplemented mice. Hepatic (A) cyclin D1, (B) cyclin E, and (C) cyclin B1 mRNA expression after partial hepatectomy (*P < 0.05).

D10 Supplementation Does Not Disrupt Cytokine Signaling During Liver Regeneration.

Activation of tumor necrosis factor alpha–interleukin-6-signal transducer and activator of transcription protein 3 signaling (STAT3), which regulates liver regeneration,13–15, 35 was examined next. This analysis showed that plasma tumor necrosis factor alpha and interleukin-6 levels were comparably induced (Supporting Fig. 2A,B), and hepatic STAT3 was similarly phosphorylated (Supporting Fig. 2C,D) after partial hepatectomy in D10-treated and control animals. Thus, D10 supplementation does not prevent cytokine signaling during liver regeneration.

D10 Supplementation and Immediate Early Gene Expression During Liver Regeneration.

Next, the immediate early gene response to partial hepatectomy was investigated. As noted previously, induction of hepatic mRNA expression of c-Fos and c-Myc and that of c-Jun, which are characteristic of this response,20, 36 were not suppressed by D10 (Supporting Fig. 3A-C). In contrast, expression of phosphoenolpyruvate carboxykinase (PEPCK), which regulates gluconeogenesis and is also part of the immediate early gene response, was suppressed by D10 supplementation before partial hepatectomy; however, its subsequent induction was not affected (Supporting Fig. 3D; *P < 0.04). Taken together, these data show that D10 does not cause global disruption of hepatic immediate early gene expression after partial hepatectomy.

D10 Supplementation Blocks Suppression of CCAAT/Enhancer Binding Protein Alpha Expression After Partial Hepatectomy.

The expression patterns of the CCAAT/enhancer binding proteins (C/EBPs) α and β are precisely regulated during liver regeneration, with C/EBPα levels declining and C/EBPβ levels increasing over the initial 24 hours after partial hepatectomy37 (Fig. 4A-F). Several observations suggest that such regulation is important for normal hepatic regeneration: For example, C/EBPβ null mice exhibit impaired liver regeneration,38 and C/EBPα is mito-inhibitory in many cell types and tissues.39 To further investigate the basis for D10-mediated inhibition of liver regeneration, the effect of dextrose on hepatic expression of these factors was evaluated. The results showed that induction of hepatic C/EBPβ mRNA expression was comparable in D10-treated and control animals (Fig. 4A). Furthermore, D10 did not inhibit induction of either the liver-enriched activator protein (LAP) or liver enriched inhibitory protein (LIP). LIP and LAP are alternative C/EBPβ translational products reported to differentially modulate liver regeneration.40 Our analysis showed modestly increased hepatic expression of liver-enriched inhibitory protein in D10-treated animals during early liver regeneration, although this difference was not statistically significant (Fig. 4B,C). In contrast, hepatic C/EBPα expression was dysregulated in D10-supplemented animals, with dextrose treatment associated with failure to suppress C/EBPα mRNA expression 0 to 24 hours after partial hepatectomy (Fig. 4D) and increased expression of the transcriptionally active 42-kDa C/EBPα protein isoform at 0 and 24 hours after surgery (Fig. 4E,F; **P < 0.02 versus water). These data raise the possibility that disrupted regulation of C/EBPα expression may contribute to the inhibitory effect of D10 on liver regeneration.

Figure 4.

C/EBP expression during liver regeneration in D10-supplemented mice. Hepatic (A) C/EBPβ and (D) C/EBPα mRNA expression at serial times after partial hepatectomy (*P < 0.05 versus 0 hours). Representative immunoblot (C, F) and summary of densitometric analysis (B, E) of C/EBP protein expression after partial hepatectomy (*P < 0.03 versus time 0 hours; **P < 0.02 versus water).

D10 Supplementation Augments p21Waf1/Cip1 and p27Kip1 Expression After Partial Hepatectomy.

The early signaling events that characterize the hepatic regenerative response, including those described previously and others, culminate in cyclin-dependent hepatocyte re-entry into and progression through the cell cycle. As part of this process, hepatocellular cyclin D1 and cyclin E expression are induced during G1 and mediate progression into S phase.41 Subsequently, cyclin B expression is induced and promotes progression through G2 into the mitotic phase. To further characterize the inhibitory effect of D10 on liver regeneration, the influence of D10 on hepatic cyclin mRNA expression was examined. The results showed that cyclin D1 and cyclin E mRNA expression were not suppressed by D10 (Fig. 5A,B). In fact, cyclin D1 was more highly expressed after partial hepatectomy in D10-treated than in control mice, although this difference did not reach statistical significance (Fig. 5A; P = 0.3). In contrast, cyclin B1 expression was significantly decreased in D10-treated mice (Fig. 5C; *P < 0.05). Taken together, these data indicate that D10 supplementation inhibits liver regeneration downstream of cyclin D1 expression but before initiation of hepatocellular DNA synthesis, thus raising the possibility that the inhibitory effect of D10 is mediated by cyclin-dependent kinase inhibitors. To investigate this possibility, the effects of D10 supplementation on expression of p21Waf1/Cip1 and p27Kip1 after partial hepatectomy were evaluated. The results showed that both mRNA and protein expression of p21 (Fig. 6A -C; *P < 0.001; **P < 0.03) and p27 (Fig. 6D-F; *P < 0.001; **P < 0.01) were increased by D10 supplementation during early regeneration, consistent with a potential functional role for these cell cycle inhibitors in mediating the inhibitory effect of D10 on liver regeneration.

Figure 6.

p21Waf1/Cip1 and p27Kip1 expression during liver regeneration in D10-supplemented mice. Hepatic (A) p21 and (D) p27 mRNA expression after partial hepatectomy (*P < 0.001 for p21 and p27, water versus D10). Representative immunoblot (C, F) and summary of densitometric analysis (B, E) of p21 and p27 protein expression after partial hepatectomy (**P < 0.03 for p21, P < 0.01 for p27, water versus D10).

D10 Supplementation Inhibits Hepatic Forkhead Box M1 Expression During Liver Regeneration.

Forkhead box transcription factor (FoxM1) is essential for normal hepatic regeneration42 and is known to suppress hepatic expression of p21 during the regenerative response.43 Therefore, the effect of D10 on hepatic FoxM1 expression during liver regeneration was investigated. The results showed that induction of FoxM1 mRNA expression after partial hepatectomy, which normally peaks 36 to 48 hours after surgery42 (Fig. 7), was significantly suppressed by dextrose administration (Fig. 7; *P < 0.04). Thus, D10 may inhibit hepatic regeneration by suppressing the induction of FoxM1 expression.

Figure 7.

FoxM1 expression during liver regeneration in D10-supplemented mice. Hepatic FoxM1 mRNA expression after partial hepatectomy (*P < 0.04).

Liver Regeneration Is Not Suppressed by D10 in p21 Null Mice.

The data described above indicate that D10 supplementation is associated with increased expression of the mito-inhibitory factors C/EBPα, p21, and p27, and decreased expression of an essential promoter of liver regeneration, FoxM1, in regenerating liver. To address the possibility that elevation of p21 expression may mediate subsequent inhibitory effects of D10 on hepatocellular cell cycle progression, the effects of dextrose on liver regeneration in p21 null mice were investigated. In this experiment, regenerating liver was harvested 36 hours after partial hepatectomy, which is the time corresponding to peak hepatocellular proliferation in p21 knockout mice.44 In contrast to wild-type mice, hepatocellular proliferation was not suppressed by D10 supplementation in these animals (Fig. 8A,B). In addition, neither FoxM1 nor cyclin B expression was inhibited by D10 treatment in p21 null mice (Fig. 8C,D). In fact, expression of each of these genes was approximately twofold higher in D10-treated compared with control animals, although these differences were not statistically significant (P = 0.2 for cyclin B; P = 0.1 for FoxM1). Taken together, these data establish that p21 is required for the inhibitory effects of D10 on FoxM1 expression and hepatocellular proliferation after partial hepatectomy.

Figure 8.

Liver regeneration in D10-supplemented p21Waf1/Cip1-null mice. (A) Immunohistochemical analysis and (B) summary of hepatocellular BrdU incorporation 36 hours after partial hepatectomy in control (water) and D10-supplemented p21-null mice (100-μm bar in upper left panel). Hepatic (C) cyclin and (D) FoxM1 mRNA expression in p21 null mice 36 hours after partial hepatectomy.

Discussion

The studies reported here elucidate the mechanisms that contribute to dextrose-mediated inhibition of hepatic regeneration, demonstrating D10-dependent increases in expression of the mito-inhibitory transcription factor C/EBPα and the cell cycle inhibitors p21 and p27, and decreased expression of FoxM1 after partial hepatectomy. These data provide in vivo evidence suggesting that early suppression of hepatic C/EBPα expression after partial hepatectomy, which is a well-known characteristic of normal liver regeneration,37 is required for a competent regenerative response. FoxM1 expression is known to suppress p21 expression during and also to be essential for normal liver regeneration.42, 43 The data reported here showing that D10-mediated inhibition of FoxM1 expression and hepatic regeneration is abrogated in p21-deficient mice indicate that p21 must also negatively regulate FoxM1 expression.

The mechanistic perturbations identified in the studies reported here are remarkably similar to those described in association with the impaired hepatic regenerative response seen in older rodents. Indeed, sustained C/EBPα expression,45 increased expression of p21,45 and decreased FoxM1 expression42, 46 are each associated with the diminished regenerative capacity of the aged rodent liver. Taken together, these observations suggest that similar perturbations in metabolic regulation after partial hepatectomy in D10-supplemented and old animals may account for the impaired regenerative response observed in each of these settings. Our data also may have broader mechanistic implications. Taken together with the central role of the liver as the principle intermediary between dietary nutrient uptake and extrahepatic energy consumption,47 the observations reported here suggest a model in which functional hepatic sufficiency is defined by homeostasis between the energy-generating capacity of the liver and the energy demands of the body mass, with liver regeneration initiated when the functional liver mass is insufficient to meet such demands. This model is consistent with a recent report suggesting that rapid, marked loss of hepatic adenosine triphosphate (ATP) after partial hepatectomy contributes to the signals that initiate liver regeneration.48

Finally, these data have potential clinical implications for hospitalized patients with acute liver injury. It is common practice in such patients to provide an intravenous dextrose infusion while monitoring serum glucose. This practice is intended to prevent morbidity associated with hypoglycemia as a result of compromised hepatic function. The data reported here highlight the possibility that this activity may have unanticipated effects on the ability of the liver to recover in these settings and suggest that studies examining the relationship between glycemic control and recovery from acute failure, partial resection, and even transplantation of small-for-size grafts may lead to improved management and outcomes in these settings.

Acknowledgements

The authors thank Drs. Paul Hruz, Lou Muglia, Eyal Shteyer, and Phillip Tarr for helpful discussions regarding these studies.

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