(66b) Effects of Glucose and Insulin Levels on HepG2 Cell Metabolism

HepG2, hepatocellular carcinoma cells, are easily maintained and expanded in culture and have been shown to express a wide range of liver-specific functions. As such, HepG2 cells can be used as an efficient system for in vitro toxicology studies and possibly in bioartificial livers (Allen et al., 2001, Park & Lee, 2005). Although the molecular expression of HepG2 cells and biological phenotypes have been characterized extensively, relatively little data exists regarding quantitative metabolic information in response to simple medium perturbations.

In this study, HepG2 cells were cultured in vitro under varying levels of glucose and insulin for five days. Four different levels of glucose were used- Minimal essential media (MEM) 0.8 mg/ml glucose (Control), DMEM-5 mg/ml glucose (high glucose), DMEM-1 mg/ml glucose (low glucose) and DMEM-0 mg/ml glucose (no glucose). Each of the four media was studied at two levels of insulin- no insulin and low insulin (50 mU/ml, representing physiological levels). Cell growth was found to be highest for DMEM-1 and lowest for DMEM-0. We also observed that the urea production was highest on a per cell basis for DMEM-0, for both no and low insulin treatments. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high glucose media.

Other measurements that were performed included amino acid uptake, fatty acid production, and albumin production. The metabolic network consisted of reactions of central metabolism such as glycolysis, pentose phosphate pathway, lactate metabolism and TCA cycle, amino acid catabolism, urea cycle, fatty acid metabolism, lipid metabolism and albumin synthesis. Using the above mentioned network, extracellular measurements, and stoichiometric material balances on each metabolite in a flux balance analysis (FBA) framework, metabolic flux maps with quantitative values of the intracellular fluxes under varying levels of glucose and insulin were evaluated. Further, by comparing the intracellular metabolic fluxes of HepG2 cells among varying treatments, alterations in central metabolic pathways have been demonstrated.

We are currently using the HepG2 in vitro culture system to study drug and toxin metabolism. Previous studies with HepG2 have demonstrated the apoptotic effect of low concentrations of ethanol and corresponding identification of signal transduction pathways involved (Castaneda & Rosin-Steiner, 2006). Another study simultaneously exposed HepG2 cells to different concentrations of acetaminophen and N-acetylcysteine (Manov et al., 2004). They showed that while N-acetylcysteine prevented oxidative damage induced by acetaminophen, it could not prevent the induced apoptosis. Interestingly in vivo studies have also shown that N-acetylcysteine is protective only when administered early on after an acetaminophen overdose (Jones, 1998). Through combinatorial exposure of HepG2 cells to acetaminophen and either protective compounds (N-acetylcysteine) or a second xenobiotic (e.g., flutamide), we seek to identify metabolic alternatives that precede irreversible hepatic injury. To this end, we have an existing pathway model of hepatic metabolism with detoxification pathways. The interactions of the xenobiotic pathways with central metabolism are analyzed using FBA enabling the identification of corresponding regulatory pathways.

References

1 Allen J.W., Hassanein T. & Bhatia S.N. (2001) Advances in bioartificial liver devices. Hepatology, 34, 447-455.

2 Castaneda F. & Rosin-Steiner S. (2006) Low concentration of ethanol induce apoptosis in HepG2 cells: role of various signal transduction pathways. International Journal of Medical Sciences., 3, 160-167.

3 Jones A.L. (1998) Mechanism of action and value of N-acetylcysteine in the treatment of early and late acetaminophen poisoning: a critical review. Journal of Toxicology - Clinical Toxicology, 36, 277-285.

4 Manov I., Hirsh M. & Iancu T.C. (2004) N-Acetylcysteine does not Protect HepG2 Cells against Acetaminophen-Induced Apoptosis. Pharmacology and Toxicology, 94, 213-225.

5 Park J.K. & Lee D.H. (2005) Bioartificial liver systems: current status and future perspective. Journal of Bioscience and Bioengineering, 99, 311-319.