Elsevier

Journal of Hepatology

Volume 60, Issue 6, June 2014, Pages 1203-1211
Journal of Hepatology

Research Article
A switch in the source of ATP production and a loss in capacity to perform glycolysis are hallmarks of hepatocyte failure in advance liver disease

https://doi.org/10.1016/j.jhep.2014.02.014Get rights and content

Background & Aims

The cause of hepatic failure in the terminal stages of chronic injury is unknown. Cellular metabolic adaptations in response to the microenvironment have been implicated in cellular breakdown.

Methods

To address the role of energy metabolism in this process we studied mitochondrial number, respiration, and functional reserve, as well as cellular adenosine-5′-triphosphate (ATP) production, glycolytic flux, and expression of glycolysis related genes in isolated hepatocytes from early and terminal stages of cirrhosis using a model that produces hepatic failure from irreversible cirrhosis in rats. To study the clinical relevance of energy metabolism in terminal stages of chronic liver failure, we analyzed glycolysis and energy metabolism related gene expression in liver tissue from patients at different stages of chronic liver failure according to Child-Pugh classification. Additionally, to determine whether the expression of these genes in early-stage cirrhosis (Child-Pugh Class A) is related to patient outcome, we performed network analysis of publicly available microarray data obtained from biopsies of 216 patients with hepatitis C-related Child-Pugh A cirrhosis who were prospectively followed up for a median of 10 years.

Results

In the early phase of cirrhosis, mitochondrial function and ATP generation are maintained by increasing energy production from glycolytic flux as production from oxidative phosphorylation falls. At the terminal stage of hepatic injury, mitochondria respiration and ATP production are significantly compromised, as the hepatocytes are unable to sustain the increased demand for high levels of ATP generation from glycolysis. This impairment corresponds to a decrease in glucose-6-phosphatase catalytic subunit and phosphoglucomutase 1. Similar decreased gene expression was observed in liver tissue from patients at different stages of chronic liver injury. Further, unbiased network analysis of microarray data revealed that expression of these genes was down regulated in the group of patients with poor outcome.

Conclusions

An adaptive metabolic shift, from generating energy predominantly from oxidative phosphorylation to glycolysis, allows maintenance of energy homeostasis during early stages of liver injury, but leads to hepatocyte dysfunction during terminal stages of chronic liver disease because hepatocytes are unable to sustain high levels of energy production from glycolysis.

Introduction

Chronic injury, mediated by a number of different etiologies, produces cirrhosis of the liver [1]. End-stage cirrhosis results in more than 30,000 deaths per year in the US, which is the 6th most frequent cause of death in individuals 25–44 years of age [2]. As liver function in cirrhosis deteriorates, patients develop jaundice, encephalopathy, an increased risk of bleeding, and muscle wasting [3]. In addition, they are susceptible to episodes of acute deterioration of hepatic function with minor precipitating events [3], [4], [5]. The mechanisms responsible for deterioration of hepatic function in cirrhosis are incompletely understood.

Metabolic adaption during environmental stress is currently an area of intense investigation because of its potential relationship to human disease [6]. Alterations in lipid and amino acid metabolism are found in patients with cholestatic liver disease and such abnormalities are associated with disease progression and hepatic failure [7], [8], [9], [10]. Thus far, however, the mechanisms responsible for these metabolomic changes have not been identified [10], [11], [12], [13], [14], [15], [16].

Oxidative phosphorylation is the major source of ATP in normal cells; however, this source of energy can change depending on microenvironment stressors [17], [18], [19], [25]. In mammalian cells, a decrease in the availability of oxygen reprograms the mitochondria to generate ATP more from glycolysis instead of oxidative phosphorylation. Recent work in cancer and other disease processes has also shown that mammalian cells can switch their source of energy production from mostly oxidative phosphorylation to mostly glycolysis and back depending on the microenvironment, genetics, epigenetic changes, and exposure to toxins [6], [17], [19], [20], [25].

Since integrity of mitochondrial function is critical for both cell survival and for the generation of new cells [21], mitochondrial dysfunction could limit the survival, function, or regeneration capacity of hepatocytes in cirrhosis. Therefore, we examined the energetics and the extent of metabolic adaptation in hepatocytes from livers at various stages of liver injury.

In the present study, we demonstrate that mitochondrial energy production remains intact during the early stages of chronic liver injury despite the fact that the number of mitochondria per hepatocyte is reduced. To maintain energy homeostasis, ATP production switches from being predominantly from oxidative phosphorylation to predominantly from glycolysis. However, maintenance of energy production by this compensatory mechanism fails in hepatocytes in later stages of chronic liver injury and is associated with hepatic failure and death.

Section snippets

Animals and chemical induced cirrhosis model

Liver cirrhosis was induced by continuous chemical treatment using phenobarbital (Sigma) and carbon tetrachloride (CCl4, Sigma) in Lewis rats as described in our previous study [22], [23]. (For detailed description please see Supplementary materials and methods.)

Isolation of rat hepatocytes and cell culture

Hepatocytes were isolated from cirrhotic and age-matched non-treated animals using a modified collagenase perfusion technique as described previously [22]. Briefly, perfusion of the portal vein using a 20G catheter (Becton, Dickinson

Animal models for liver cirrhosis

Hepatocytes were isolated from the livers of animals (rats) with cirrhosis and normal liver function, hereafter denoted as “hepatocytes from early cirrhotic livers” and from the livers of animals (rats) with cirrhosis and sustained loss of liver function 4 weeks after they received their last dose of carbon tetrachloride, hereafter denoted “hepatocytes from failing cirrhotic livers” (Supplementary Fig. 1) [22], [23]. All experiments were performed on isolated hepatocytes rather than liver tissue

Discussion

There is an abundant literature on the identification of mechanisms responsible for development of cirrhosis, but little concerning the mechanisms responsible for organ failure in terminal chronic liver disease. The present study was conducted to determine whether alterations in energy production and utilization could be linked to hepatocyte dysfunction in cirrhosis. Using a unique rat model of cirrhosis and end-stage liver failure that resembles human disease, we demonstrate that (a)

Financial support

This work was supported by grants from NIH, DK48794 and DK09932 to I.J.F.; DK083556 to A.S.-G.; AG034995 to I.I.P.; and DK090325 to M.O.; U54-CA112970 to P.T.R. and Rice University Start Up to D.N. The CCBTP training grant from the CPRIT funded V.S. and the Odessy Fellowship (MDACC) funded T.M.

Conflict of interest

The authors who have taken part in this study declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Authors’ contribution

Study concept and design: A.S.-G., I.J.F., D.N.; acquisition of data: T.N., N.B., A.D., H.B., Z.Z., K.H., M.I.Y., M.O., I.P., A.S.-G., D.N., I.J.F.; analysis and interpretation of data: T.N., N.B., A.D., A.S.-G., N.B., D.N., I.J.F.; microarray analysis: V.S., T.J.M., P.R.; drafting of the manuscript: T.N., N.B., A.S.-G., D.N.; critical revision of the manuscript for intellectual content: T.N., N.B., I.P., A.S.-G., I.J.F., D.N.; obtained funding: A.S.-G., I.J.F., I.P., D.N.; administrative,

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    These authors contributed equally to this work.

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