Metabolic pre‐conditioning of cultured cells in physiological levels of insulin: Generating resistance to the lipid‐accumulating effects of plasma in hepatocytes

Abstract

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.

Metabolic flux analysis of cultured hepatocytes exposed to plasma

“…We previously found that preconditioning rat hepatocyte cultures in low-insulin-containing medium prior to plasma exposure decreased the storage of TG, whereas supplementing the plasma cultures with amino acids restored albumin and urea synthesis as well as ammonia removal rate (Chan et al, 2002). In what follows we investigate the metabolic and functional adaptations associated with plasma exposure from insulin preconditioning and amino acid supplementation using MFA to yield both a qualitative and quantitative measure of the changes in the internal workings of hepatocyte metabolism.…”

“…More recently, we noted that preconditioning primary rat hepatocytes in culture medium containing low levels of insulin reduced intracellular lipid accumulation during subsequent plasma exposure (Chan et al, 2002), and that amino acid supplementation to the plasma restored albumin and urea secretion, as well as ammonia removal (Chan et al, 2002;Washizu et al, 2000). In the current study, we use an MFA model to investigate the effects of insulin and amino acids on intracellular metabolic fluxes of primary rat hepatocytes.…”

Integration of Technologies for Hepatic Tissue Engineering

“…Supraphysiological levels of hormones can also lead to paradoxical responses when cultured hepatocytes are placed in animal or human plasma, clearly a more physiologically relevant fluid than culture medium. Prior studies show that rat hepatocytes become severely fatty and lose hepatic functions when transferred from culture medium to plasma [98], but that plasma-induced intracellular lipid accumulation can be eliminated if culture medium containing low insulin levels is used prior to exposure to plasma [99].…”

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

“…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.…”