A method for the determination of glucose synthesis in isolated bovine hepatocytes.
A simple method for determining glucose synthesis from radiolabeled precursors in isolated bovine hepatocytes using ion exchange resins is presented. This method allows processing of multiple small volume samples using suspensions of anion and cation exchange resins rather than traditional stacked column separation methods. Hepatocytes were isolated from calf liver by collagenase perfusion of the caudate lobe and were incubated with (14)C-labeled lactate or propionate as gluconeogenic substrates. Glucose synthesis was determined in an aliquot of cell suspension that was vortexed with a slurry of anion exchange (acetate form) resin, followed by a slurry of cation exchange resin. Newly synthesized, labeled glucose was recovered in the supernatant after centrifugation and quantitated by scintillation counting. Using this procedure, more than 98% of the unused labeled precursor was bound to the ion exchange resin and essentially 100% of a labeled glucose tracer was recovered in the supernatant. Pretreatment of hepatocyte suspensions with glucose oxidase was shown to eliminate the accumulation of radioactivity in the supernatant, thus confirming the specificity of this technique for measurement of newly synthesized glucose. This method was sensitive to changes in the rate of hepatic gluconeogenesis that resulted from changes in substrate concentration or the addition of glucagon or fatty acids to the hepatocyte incubations.
Azain
M J
MJ
Animal and Dairy Science Department, University of Georgia, Athens, GA 30602, USA.
Kasser
T R
TR
Baile
C A
CA