A method for the measurement of lactate, glycerol and fatty acid production from 14C-glucose in primary cultures of rat epididymal adipocytes.

Abstract

We have developed a method for the analysis of the main metabolic products of utilization of glucose by isolated adipocytes. They were incubated for 24 h with 14C-glucose. The final label distribution and cold levels of medium glucose, lactate and glycerol were estimated. Medium lactate was extracted using ion-exchange resin minicolumns prepared with centrifugation-filtering tubes in which the filter was substituted by the resin. This allowed complete washing using only 0.2 mL. Repeated washings allowed for complete recovery of fractions with low volumes passing through or retained (and eluted), which permitted precise counting and a sufficient amount of sample for further analyses. Lactate was separated from glucose and glycerol; glucose was then separated by oxidizing it to gluconate with glucose oxidase, and glycerol was separated in parallel by phosphorylation with ATP and glycerol kinase. Cells' lipid was extracted with ether and saponified. Glycerides-glycerol and fatty acids (from the soaps) were counted separately. The complete analysis of cells incubated with labelled glucose resulted in about half of the glucose metabolized in 24 h, 2/3rds of the incorporated glucose label was found as lactate, and 14% as free glycerol. Their specific activities per carbon were the same as that of glucose. Production of fatty acids took about 5% of the label incorporated, an amount similar to that of glycerides-glycerol and estimated carbon dioxide. The procedure described is versatile enough to be used under experimental conditions, with a high degree or repeatability and with only about 3% of the label not accounted for.