A method for the determination of glucose synthesis in isolated bovine hepatocytes

A simple method for determining glucose synthesis from radiolabeled precurs ors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspen sions of anion and cation exchange resins rather than traditional stacked c olumn separation methods. Hepatocytes were isolated from calf liver by coll agenase perfusion of the caudate lobe and were incubated with C-14-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis,vas de termined in an aliquot of cell suspension that was vortexed with a slurry o f anion exchange (acetate form) resin, followed by a slurry of cation excha nge resin. Newly synthesized, labeled glucose was recovered in the supernat ant after centrifugation and quantitated by scintillation counting. Using t his procedure, move than 98% of the unused labeled precursor was bound to t he ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with g lucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for meas urement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substr ate concentration or the addition of glucagon or fatty acids to the hepatoc yte incubations. (C) Elsevier Science Inc. 1999. All rights reserved.