Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells.

Gabriel Yarmush,Francois Berthiaume,Mubasher Saleem,Rene Schloss,Srivathsan Koundinyan,Timothy Maguire,Joshua Yarmush,Martin Yarmush,Lucas Santos,Nir Nativ

doi:10.3390/metabo6010001

Abstract

Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

Highlights

IntroductionWith over 15,000 patients on the waiting list for liver donations and approximately 7000 liver transplants conducted annually, 2000 patients die each year [1] of acute and chronic liver failure
With over 15,000 patients on the waiting list for liver donations and approximately 7000 liver transplants conducted annually, 2000 patients die each year [1] of acute and chronic liver failure.Hepatic macrosteatosis, defined as abnormal lipid accumulation in hepatocytes in the form of large lipid droplets, predisposes to primary non-function following transplantation and is one of the most common causes for a donor organ removal from the donor pool [2]
HepG2 cells have been previously shown to accurately depict hepatic metabolic function [15,16,17,18,19,20], and were used as a surrogate system to evaluate the effects of steatosis and subsequent defatting on liver metabolism

Summary

Introduction

With over 15,000 patients on the waiting list for liver donations and approximately 7000 liver transplants conducted annually, 2000 patients die each year [1] of acute and chronic liver failure. Hepatic macrosteatosis, defined as abnormal lipid accumulation in hepatocytes in the form of large lipid droplets, predisposes to primary non-function following transplantation and is one of the most common causes for a donor organ removal from the donor pool [2]. Pathologic analysis is conducted on all donated livers, and if more than 30% of the hepatocytes appear to be macrosteatotic, the liver is generally discarded from the donor pool. If all steatotic livers could be made suitable for transplantation, more than 1000 viable livers could be added to the donor pool annually, resulting in sufficient transplants that could halve the number of patients dying while on the waiting list [3].

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Results

Conclusion