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

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-alpha) 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/EBPbeta), but did augment expression of the mito-inhibitory factors C/EBPalpha, p21(Waf1/Cip1), and p27(Kip1). 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/EBPalpha, 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.

Figure 1. Liver Regeneration in D10-Supplemented Mice

(A) Immunohistochemical analysis of hepatocellular BrdU incorporation (upper panels) and histological analysis (H&E, lower panels) of liver 36 hours after partial hepatectomy in control (water) and dextrose (D10)-supplemented mice (100 micron bar in upper left panel). (B) Summary of hepatocellular proliferation (fraction of total hepatocytes that incorporated BrdU) 24–72 hours after partial hepatectomy in regenerating liver from control and D10-supplemented mice (*p<0.02). (C) H&E 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).

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 0hr). (D) Representative protein immunoblot for hepatic phosphorylated and total p42/44 ERK after partial hepatectomy. (E) TGFα mRNA expression and (F) representative TGFα protein immunoblot after partial hepatectomy (*p<0.02 versus 0hr).

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 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 hr). 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 hr; **p<0.02 versus water).

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).

Figure 6. p21^Waf1/Cip1 and p27^Kip1 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).

Figure 7. FoxM1 Expression during Liver Regeneration in D10-Supplemented Mice

Hepatic FoxM1 mRNA expression after partial hepatectomy (*p<0.04).

Figure 8. Liver Regeneration in D10-Supplemented p21^Waf1/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 micron bar in upper left panel). Hepatic (C) cyclin and (D) FoxM1 mRNA expression in p21 null mice 36 hours after partial hepatectomy.