Adenine nucleotide changes in the remnant liver: An early signal for regeneration after partial hepatectomy

Abstract

Liver regeneration after partial hepatectomy (PHx) is orchestrated by multiple signals from cytokines and growth factors. We investigated whether increased energy demand on the remnant liver after PHx contributes to regenerative signals. Changes in the tissue's energy state were determined from adenine nucleotide levels. Adenosine triphosphate (ATP) levels in remnant livers decreased markedly and rapidly (to 48% of control by 30 seconds post-PHx) and remained significantly lower than those in sham-operated controls for 24 to 48 hours. The ATP decrease was not reflected in corresponding increases in adenosine diphosphate (ADP) and adenosine monophosphate (AMP), resulting in a marked decline in total adenine nucleotides (TAN). We found no evidence of mitochondrial damage or uncoupling of oxidative phosphorylation. Multiple lines of evidence indicated that the decline in TAN was not caused by increased energy demand, but by ATP release from the liver. The extent of ATP loss was identical after 30% or 70% PHx, whereas fasting or hyperglycemia, conditions that greatly alter energy demand for gluconeogenesis, affected the ATP/ADP decline but not the loss of TAN. Presurgical treatment with the alpha-adrenergic antagonist phentolamine completely prevented loss of TAN, although changes in ATP/ADP were still apparent. Importantly, phentolamine treatment inhibited early signaling events associated with the priming stages of liver regeneration and suppressed the expression of c-fos. Pretreatment with the purinergic receptor antagonist suramin also partly suppressed early regenerative signals and c-fos expression, but without preventing TAN loss.

Conclusion: The rapid loss of adenine nucleotides after PHx generates early stress signals that contribute to the onset of liver regeneration.

Fig 1. Adenine nucleotide changes in remnant livers following 70% PHx or Sham surgery

Livers from PHx (-●-) or Sham (-○-) operated rats were freeze-clamped at designated post-operative times and processed for adenine nucleotide analysis. (A) ATP; (B) ADP; (C) AMP; (D) Total adenine nucleotides, TAN; (E) ATP/ADP. For estimates of statistical significance, PHx and Sham livers from 4–13 animals were compared for each time point (see supplemental Table S1); ** p<0.01, * p<0.05.

Fig 1. Adenine nucleotide changes in remnant livers following 70% PHx or Sham surgery

Livers from PHx (-●-) or Sham (-○-) operated rats were freeze-clamped at designated post-operative times and processed for adenine nucleotide analysis. (A) ATP; (B) ADP; (C) AMP; (D) Total adenine nucleotides, TAN; (E) ATP/ADP. For estimates of statistical significance, PHx and Sham livers from 4–13 animals were compared for each time point (see supplemental Table S1); ** p<0.01, * p<0.05.

Fig 2. Lactate/pyruvate ratios (L/P) and β-hydroxybutyrate/acetoacetate (BHB/AcAc) ratios in remnant livers following 70% PHx or Sham surgery

Metabolite levels were analyzed in neutralized extracts from remnant livers of PHx and Sham operated rats. A) L/P ratios for PHx (-●-) and Sham (-○-); B) BHB/AcAc ratios for PHx (-■-) and Sham (-□-). For estimates of statistical significance, PHx and Sham livers from 3–7 animals were compared for each time point (see supplemental Table S4); ** p<0.01, * p<0.05.

Fig 3. Blood glucose levels after 70% PHx and Sham surgery

Fed PHx rats (-○-); 24 h fasted PHx (-△-) rats; fed Sham-operated rats (-●-); phentolamine treated (10 mg/kg, 30 min prior to surgery) PHx rats (-□-). Error bars indicate SEM for 3–5 animals for each condition. Where not shown, standard errors were less than the symbol size.

Fig 4. Effect of circulating blood glucose on adenine nucleotide changes after PHx

(A) ATP content, (B) ATP/ADP, (C) total adenine nucleotide content, 0–15 min following 70% PHx, under euglycemic, hypoglycemic and hyperglycemic conditions. Data obtained from 3–5 rats per treatment for each time point. ** p<0.01, * p<0.05, comparing PHx and Sham-operated animals.

Fig 5. Effect of phentolamine treatment on adenine nucleotide changes after PHx

(A) ATP content, (B) ATP/ADP ratio, (C) total adenine nucleotide content, 0–15 min following 70% PHx in phentolamine treated rats (10 mg/kg, 30 min prior to surgery). Data represent 3–5 rats per treatment for each time point. ** p<0.01, * p<0.05, comparing phentolamine-treated and control animals.

Fig 6. Effect of phentolamine and suramin treatment on JNK phosphorylation and c-fos expression in remnant livers after PHx

(A) Western blots of phospho-JNK(p54) and JNK(p54) protein levels in liver extracts from untreated animals that did not undergo surgery (lanes 1–2) and control (lanes 3–5), phentolamine-treated (lanes 6–8), or suramin-treated (lanes 9–11) animals subjected to PHx (upper blots) or Sham (lower blots) surgery. Inhibitors were injected ip 30 min prior to surgery and tissues were harvested 30 min after surgery. Control animals received a corresponding volume of saline. (B) Effect of phentolamine or suramin treatment on JNK phosphorylation following PHx or Sham surgery. Densities obtained from Western blots shown in (A) for phospho-JNK were normalized to total JNK protein in each sample,. ** p<0.01. (C) Effect of phentolamine or suramin treatment on c-fos relative to GAPDH mRNA by quantitative RT-PCR, expressed as ΔΔCt on a ₂log scale comparing samples from PHx and Sham surgeries. Samples were obtained 30 min after PHx or Sham surgery using 3 animals for each condition; ** p<0.01, * p<0.05.