Postponing the Hypoglycemic Response to Partial Hepatectomy Delays Mouse Liver Regeneration

Abstract

All serious liver injuries alter metabolism and initiate hepatic regeneration. Recent studies using partial hepatectomy (PH) and other experimental models of liver regeneration implicate the metabolic response to hepatic insufficiency as an important source of signals that promote regeneration. Based on these considerations, the analyses reported here were undertaken to assess the impact of interrupting the hypoglycemic response to PH on liver regeneration in mice. A regimen of parenteral dextrose infusion that delays PH-induced hypoglycemia for 14 hours after surgery was identified, and the hepatic regenerative response to PH was compared between dextrose-treated and control mice. The results showed that regenerative recovery of the liver was postponed in dextrose-infused mice (versus vehicle control) by an interval of time comparable to the delay in onset of PH-induced hypoglycemia. The regulation of specific liver regeneration-promoting signals, including hepatic induction of cyclin D1 and S-phase kinase-associated protein 2 expression and suppression of peroxisome proliferator-activated receptor γ and p27 expression, was also disrupted by dextrose infusion. These data support the hypothesis that alterations in metabolism that occur in response to hepatic insufficiency promote liver regeneration, and they define specific pro- and antiregenerative molecular targets whose regenerative regulation is postponed when PH-induced hypoglycemia is delayed.

Supplemental Figure S1

Delaying partial hepatectomy (PH)-induced hypoglycemia alters glycogen synthase kinase (GSK)3β and Akt phosphorylation. Equal quantities of protein from individual replicate samples of whole liver lysate for each experimental condition were pooled and subjected to SDS-PAGE and immunoblot analyses for hepatic levels of phosphorylated (p-) and total GSK3β, Akt, and cAMP response element–binding protein (Creb).

Figure 1

Delaying partial hepatectomy (PH)-induced hypoglycemia postpones regenerative hepatocellular proliferation. A: Blood glucose values before and after PH in dextrose- and vehicle-treated mice (controls). Blood glucose concentrations in all controls after PH and in dextrose-treated mice at 18 and 22 to 60 hours after PH are significantly lower than those in unoperated (ie, 0-hour) mice. Blood glucose values in dextrose-treated mice at 2 to 14 hours and 18 hours after PH are significantly different from those in corresponding control mice at those time points. The strain-specific, nonfasting, normal blood glucose of male C57BL6/J mice is 198 ± 14 mg/dL, as reported in the Mouse Phenome Database (Jackson Laboratory). B: Plasma insulin and glucagon. C and D: Hepatocellular bromodeoxyuridine (BrdU). E: Liver/body mass ratio. F: Representative hematoxylin and eosin–stained liver sections from dextrose-infused and control mice. Data are expressed as means ± SEM. n = 3 to 8 replicates per time point and treatment group. ^∗P < 0.05 versus control (B) or corresponding control (C and E); ^∗∗P < 0.01, ^∗∗∗P < 0.001 versus corresponding control; ^†P < 0.05, ^††P < 0.01, and ^†††P < 0.001 versus 0 hour; ^‡P < 0.05 versus corresponding control and 24-hour dextrose; ^‡‡P < 0.01 versus 24-hour control; ^§§§P < 0.001 versus 12-hour control. Scale bars = 100 μm.

Figure 2

Postponing partial hepatectomy (PH)-induced hypoglycemia delays the metabolic response to liver resection–induced hepatic insufficiency. A: Body mass (indexed to initial body mass). B: Serum amino acid levels. C: Serum free fatty acid levels. P = 0.05 versus control. D: Hepatic triglyceride levels. Data are expressed as means ± SEM. n = 3 to 8 replicates per time point per treatment group. ^∗P < 0.05, ^∗∗P < 0.01 versus control; ^†††P < 0.001 versus 12-hour dextrose; ^‡‡P < 0.01 versus vehicle control. Aaa, α-NH₂-adipic acid; Aab, α-NH₂-butyrate.

Figure 3

Delaying partial hepatectomy (PH)-induced hypoglycemia alters glycogen synthase kinase (Gsk)3β and Akt phosphorylation. Protein immunoblot analyses (A) and corresponding quantification (B) of hepatic levels of phosphorylated (p-) and total Gsk3β, Akt, and cAMP response element–binding protein (Creb). Data are expressed as means ± SEM. n = 4 replicates per time point per treatment group. ^∗P < 0.05 versus corresponding control.

Figure 4

Glycemic regulation of regenerative hepatic cyclin expression. Hepatic cyclin D1 mRNA (A) and protein (B and C) expression. D: Representative IHC analysis of hepatic cyclin D1 expression. E: Hepatic mRNA expression of cyclins E1, A2, and B1. Data are expressed as means ± SEM. n = 3 to 8 replicates per time point per treatment group; the immunoblot in B shows equal quantities of protein pooled from replicates for each time point and treatment group. ^∗P < 0.05, ^∗∗P < 0.01 versus control. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

Partial hepatectomy (PH)-induced hypoglycemia regulates regenerative cyclin D1 gene acetylation. A: Histone H3 acetylated on K9 (Ac-H3K9) real-time quantitative chromatin immunoprecipitation PCR (ChIP-qPCR) targeting the cyclin D1 gene (Ccnd1) sequence indicated by the gray bar in B. B: The University of California at Santa Cruz (UCSC) mm10 Ccnd1 gene map is shown above data depicting the abundances of histone acetylated sequences at 12 hours after PH in dextrose-infused and control mice. Below those data, the white bar with black border indicates sequences identified as hyperacetylated at 12 hours after PH versus sham surgery from our recently published study; the grey bar identifies the sequence targeted for qPCR in A; and the black bar indicates sequences significantly deacetylated at 12 hours after PH in regenerating liver from dextrose-infused versus control mice (q < 0.1) (Supplemental Table S1). Data are expressed as mean ± SEM percentages to input. n = 3 to 6 mice per group. ^∗P < 0.05 versus control.

Figure 6

Metabolic regulation of hepatic cyclin-dependent kinase inhibitor, S-phase kinase-associated protein (Skp)2, and peroxisome proliferator-activated receptor γ (Pparγ) expression during partial hepatectomy (PH)-induced liver regeneration. A–C: Hepatic p21 and p27 mRNA (A) and p27 protein (B and C). D: Skp2 mRNA. E–G: Pparγ1 and Pparγ2 mRNA (E) and PPARγ2 protein (F and G). n = 3 to 8 mice per time point per group and indexed to expression in 12-hour controls; n = 4 (B, C, F, G, replicates). ^∗P < 0.05, ^∗∗P < 0.01 versus control. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

A metabolic model of liver regeneration. Candidate mechanisms linking metabolism and regeneration suggested by the new data reported here and by previously published data, , are indicated by dashed and solid arrows, respectively. PPARγ, peroxisome proliferator-activated receptor gamma; Skp2, S-phase kinase-associated protein 2.