Metabolic flux analysis of cultured hepatocytes exposed to plasma

Abstract

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.

Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease

“…Furthermore, the ability of iHepatocytes2322 for performing gluconeogenesis was demonstrated using experimentally measured secretion rates for glucose and albumin, and uptake rates for glycerol, lactate, amino acids and FAs in primary rat hepatocytes 24 (Supplementary Data 6).…”

Integrated Energy and Flux Balance Based Multiobjective Framework for Large-Scale Metabolic Networks

“…Lastly, we obtained the Pareto solutions in the presence of measurement constraints. The experimentally measured flux data for gluconeogenic and glycolytic state were taken from Chan et al, 5,6 respectively.…”

“…These assumptions are discussed in detail elsewhere. [5][6][7][8] Energy Balance Analysis Energy Balance Analysis imposes constraints based on law of thermodynamics on the cellular reaction networks. 2,3 For any reaction set, if stoichiometry is represented by matrix S, l denotes an M-dimensional vector of chemical potentials, Dl denotes the N-dimensional vector of reaction potentials, then these potentials can be computed as Dl ¼ S T l. The null space matrix of S (for r linearly independent rows, with r N) is denoted by K and forms a basis for the null space of S, so that SK = 0.…”

“…1,[5][6][7][8][11][12][13][14]24,25 Flux balance analysis (FBA) uses stoichiometric and mass balance constraints to compute the intracellular fluxes. Recently, energy balance analysis (EBA) was developed to ensure the thermodynamic feasibility of the computed fluxes.…”

Integrating Gene Expression and Metabolic Profiles

“…This interval is considered the preconditioning period. The 6-day-old sandwiched hepatocyte cultures were subsequently exposed to unsupplemented or amino acid-supplemented plasma solution for an additional 7 days (11,12). The four combinations of preconditioning and plasma supplementation evaluated were as follows.…”