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

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.

Manipulation of Lipid Metabolism During Normothermic Machine Perfusion: Effect of Defatting Therapies on Donor Liver Functional Recovery

“…Urea is produced by the liver and results from an enzymatic reaction involving carbon dioxide and the ammonia derived from the deamination of proteins in the liver. The increased level of urea suggests increased urea synthesis in defatted livers . Our previously published viability criteria assert that lactate levels <2.5 mmol/L within 4 hours is a major criterion for safe transplantation of ECD donor livers.…”

“…Previous in vitro studies with fat-laden rat hepatocytes and hepatoblastoma cells demonstrated the feasibility of defatting cells with a combination of drugs over 48 hours. (6,18,19) Thereafter, this defatting cocktail was delivered to whole steatotic rat livers during NMP, and the authors demonstrated a decrease in T-TG of 50% within 3 hours. (6) Interestingly, a decrease of 30% could be obtained with NMP alone over the same period.…”

“…The increased level of urea suggests increased urea synthesis in defatted livers. (19,26) Our previously published viability criteria assert that lactate levels <2.5 mmol/L within 4 hours is a major criterion for safe transplantation of ECD donor livers. However, it also considers other parameters of liver metabolism.…”

Ex situ machine perfusion as a tool to recondition steatotic donor livers: Troublesome features of fatty livers and the role of defatting therapies. A systematic review.

“…The same protocol was later applied to human hepatoma cells (HepG2 cells) by Yarmush et al They found that the cells' lipid content decreased if left in an environment free of FAs. Analysis of metabolites present in the culture media showed that this defatting process was attributed to increased mitochondrial β‐oxidation through upregulation of the tricarboxylic acid and urea cycles.…”

Clinical Hepatocyte Transplantation: What Is Next?

“…Additional strategies such as machine perfusion techniques and perfusate selection have been shown to dramatically increase cell yields (52, 53). Moreover, machine perfusion organ reconditioning has proven valuable in improving cell viability and function of ischemic livers and experimentally on fatty livers (53–56). Alternatively, the addition of polyethylene glycol to hypothermic preservation solutions has been shown to improve hepatocyte survival following successful isolation (57–59).…”