Upregulation of Krebs cycle and anaerobic glycolysis activity early after onset of liver ischemia

<div><p>The liver is a highly vascularized organ receiving a dual input of oxygenated blood from the hepatic artery and portal vein. The impact of decreased blood flow on glucose metabolism and how hepatocytes could adapt to this restrictive environment are still unclear. Using the left portal vein ligation (LPVL) rat model, we found that cellular injury was delayed after the onset of liver ischemia. We hypothesized that a metabolic adaptation by hepatocytes to maintain energy homeostasis could account for this lag phase. Liver glucose metabolism was characterized by ¹³C- and ¹H-NMR spectroscopy and analysis of high-energy metabolites. ALT levels and caspase 3 activity in LPVL animals remained normal during the first 12 h following surgery (<i>P</i><0.05). Ischemia rapidly led to decreased intrahepatic tissue oxygen tension and blood flow (<i>P</i><0.05) and increased expression of Hypoxia-inducible factor 1-alpha. Intrahepatic glucose uptake, ATP/ADP ratio and energy charge level remained stable for up to 12 h after ligation. Entry of glucose in the Krebs cycle was impaired with lowered incorporation of ¹³C from [U^-13C]glucose into glutamate and succinate from 0.25 to 12 h after LPVL. However, total hepatic succinate and glutamate increased 6 and 12 h after ischemia (<i>P</i><0.05). Glycolysis was initially reduced (<i>P</i><0.05) but reached maximum ¹³C-lactate (<i>P</i><0.001) and ¹³C-alanine (<i>P</i><0.01) enrichments 12 h after LPVL. In conclusion, early liver homeostasis stems from an inherent ability of ischemic hepatocytes to metabolically adapt through increased Krebs cycle and glycolysis activity to preserve bioenergetics and cell viability. This metabolic plasticity of hepatocytes could be harnessed to develop novel metabolic strategies to prevent ischemic liver damage.</p></div

Time course of total and ¹³C-enriched hepatic lactate and alanine in the ligated lobe following LPVL.

<p>Unlabeled levels of hepatic (A) lactate and (B) alanine of sham-operated rats (open white squares) and LPVL-treated (filled grey squares) rats. ¹³C-enrichment of metabolites: lactate (A) and alanine (B) in sham (white patterned squares) and LPVL-treated (black patterned squares) rats. Values are expressed as the mean ± SEM of 3–5 animals per group. (**<i>P</i><0.01, ***<i>P</i><0.001 when total lactate and alanine levels were compared in (A-B)).</p

Time course of total and ¹³C glucose levels during hypoxia.

<p>(A) Total levels of hepatic glucose of sham-operated (filled white circles) and LPVL-treated (filled grey circles) rats. (B) Levels of ¹³C glucose in sham (filled white circles) versus LPVL-treated (filled grey circles) rats. [U^-13C] glucose was infused for 45 min after the indicated times. (C) Representative microphotographs of Hypoxia inducible factor 1-α (HIF 1-α) staining of the left liver lobe of LPVL-treated animals 12h after ischemia. Slides were stained with Mayer’s Hematoxylin and mouse-anti-HIF-1α. Values are expressed as the mean ± SEM of 3–10 animals per group.</p

ATP/ADP ratio and energy charge levels in the left lateral lobe of sham and LPVL-treated rats.

<p>Hepatic (A) AMP, (B) ADP, (C) ATP, (D) ATP/ADP ratio and (E) energy charge of sham and LPVL-operated rats over a period ranging from 0.25 to 12 h following ischemia. Values are expressed as the mean ± SEM of 3–8 animals.</p

Delayed hepatocyte injury after induction of ischemia.

<p>(A) Evaluation of ALT serum levels and (B) quantification of caspase 3 activity in sham and LPVL-treated rats over a period of up to 48 h following induction of ischemia. (C) Representative microphotographs of HPS staining of the left liver lobe of LPVL-operated rats over a period ranging from 6 to 48 h following ischemia. Liver injury was assessed by the histological evaluation of necrosis. Values are ±SEM of 3–8 different animals. (*<i>P</i><0.05, ***<i>P</i><0.001).</p

Time course of total and ¹³C-enriched hepatic glutamate and succinate in the ligated lobe following LPVL.

<p>Unlabeled levels of hepatic (A) glutamate and (B) succinate of sham-operated rats (open white squares) and LPVL-treated (filled grey squares) rats. ¹³C-enrichment of the metabolites: (A) glutamate and (B) succinate in sham (white patterned squares) and LPVL-treated (black patterned squares) rats. Values are expressed as the mean ± SEM of 3–6 animals per group. (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 when total glutamate and succinate levels were compared in (A-B)).</p