A method for the measurement of lactate, glycerol and fatty acid production from¹⁴C-glucose in primary cultures of rat epididymal adipocytes

Abstract

We developed a method for the analysis of the main metabolic products of utilization of glucose by isolated adipocytes. They were incubated 24 h with 14 C-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 washings using only 0.2 mL. Repeated washings allowed for complete recovery of fractions with low volumes passing through or retained (and eluted), which allowed precise counting and sufficient 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, 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, a similar amount to that of glyceridesglycerol 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.

Use of14C-glucose by primary cultures of mature rat epididymal adipocytes. Marked release of lactate and glycerol, but limited lipogenesis in the absence of external stimuli

“…Specific activities were expressed in Bq/mmol-C i.e. per micromole of the substrate divided by the number of C in the molecule, thus allowing a direct comparison of specific activities between molecules with different number of C atoms [26]. The initial incubation medium containing 7 mM glucose had a specific radioactivity of 141 Bq/mmol glucose (23.5 Bq/mmol-C) that for 14 mM was 71 Bq/mmol glucose (11.8 Bq/mmol-C).…”

“…The incubation of adipocytes was stopped by harvesting the cells after the medium was extracted, mixed, aliquoted and frozen. The procedure for measuring label distribution in the different fractions of cells and media have been previously developed, tested and quantified [26]. Briefly, the cells of wells incubated with labelled glucose were weighed, frozen with liquid nitrogen, transferred to glass tubes and immediately extracted with chilled peroxide-free diethyl ether, since it is non-reactive, and is highly effective for TAG [50].…”

“…The ether-insoluble potassium soaps were extracted and counted. The aqueous phase contained all glycerides-glycerol; it was also removed and counted [26]. Soap label was that of TAG fatty acids.…”

“…In them, most cell content is essentially restricted to a thin layer of 'live' cytoplasm between the cell membrane and the border of the huge lipid vacuole [25], far from oxidative mitochondria. Thus, in addition to hamper (oxidative) energy production from glucose, lipogenesis is also severely reduced [12,26]. Cell size, thus, has a deep influence on adipocyte function.…”

“…The present study is part of a wider effort to understand better why mature adipocytes rely so much on 3C metabolite efflux via glycolysis in detriment of lipogenesis. We have already published two papers describing the methodology used here [25,26] and distributed the final study in two papers, the first centered on how glycerol is produced and released [12] and this one, focused on the quantitative utilization of glucose to yield essentially lactate and glycerol, as well as fueling lipogenesis. Our objective was to establish a basis for the quantification of the products of adipocyte lipogenesis, and the relative importance of 3C metabolite production under basal normoxic conditions, i.e.…”