Abstract
The liver plays a key role in maintaining physiological homeostasis and hepatocytes are largely responsible for this. The use of isolated primary hepatocytes has become an essential tool for the study of nutrient physiology, xenobiotic metabolism and several liver pathologies. Since hepatocytes are removed from their normal environment, the isolation procedure and in vitro culture of primary hepatocytes is partially known to induce undesired metabolic changes. We aimed to perform a thorough metabolic profiling of primary cells before, during and after isolation using state-of-the-art techniques. Extensive metabolite measurements using HPLC were performed in situ in the liver, during hepatocyte isolation using the two-step collagenase perfusion method and during in vitro cell culture for up to 48 hours. Assessment of mitochondrial respiratory capacity and ATP-linked respiration of isolated primary hepatocytes was performed using extracellular flux analysis. Primary hepatocytes displayed a drastic decrease in antioxidative-related metabolites (NADPH, NADP, GSH and GSSG) during the isolation procedure when compared to the in situ liver (P<0.001). Parallel assessment of citric acid cycle activity showed a significant decrease of up to 95% in Acetyl-CoA, Isocitrate/Citrate ratio, Succinate, Fumarate and Malate in comparison to the in situ liver (P<0.001). While the levels of several cellular energetic metabolites such as Adenosine, AMP, ADP and ATP were found to be progressively reduced during the isolation procedure and cell culture (P<0.001), higher ATP/ADP ratio and energy charge level were observed when primary cells were cultured in vitro compared to the in situ liver (P<0.05). In addition, a significant decrease in the respiratory capacity occurred after 24 hours in culture. Interestingly, this was not associated with a significant modification of ATP-linked respiration. In conclusion, major metabolic alterations occur immediately after hepatocytes are removed from the liver. These changes persist or increase during in vitro culture. These observations need to be taken into account when using primary hepatocytes for the study of metabolism or liver physiopathology.
Highlights
The liver is central to the homeostatic maintenance of physiological metabolism
This decrease of up to 95% was observed during the earliest steps of the isolation procedure, collagenase perfusion, where both NADPH and NADP contents are reduced significantly (P
Primary cultured hepatocytes showed a reduction of 79% of the GSH/GSSG ratio compared to the in situ liver (P
Summary
The liver is central to the homeostatic maintenance of physiological metabolism. Among its many functions, it is involved in the metabolism of carbohydrates with gluconeogenesis, glycogenolysis and glycogenesis and in the metabolism of lipids with the synthesis and degradation of triglycerides, of cholesterol into bile acids as well as in lipoprotein synthesis. Several experimental models for the study of hepatocyte physiology, pharmacokinetics of drugs and pathogenesis of liver diseases have been developed and many of those rely on the use of isolated primary hepatocytes [3,4,5]. These experimental models, used in both fundamental research and pharmacological studies, were developed based on the assumption that isolated hepatocytes shared similar metabolic profiles as the ones of hepatocytes in situ [6]. The original descriptions of theses similarities were made decades ago based on data acquired using tools and techniques available at the time [7,8,9,10]
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