White adipose tissue (WAT) produces large amounts of lactate and glycerol from glucose. We used mature epididymal adipocytes to analyse the relative importance of glycolytic versus lipogenic glycerol in adipocytes devoid of external stimuli. Cells were incubated (24/48 h) with 7/14 mM glucose; half of the wells contained 14C-glucose. We analysed glucose label fate, medium metabolites, and the expression of key genes coding for proteins controlling glycerol metabolism. The effects of initial glucose levels were small, but time of incubation increased cell activity and modified its metabolic focus. The massive efflux of lactate was uniform with time and unrelated to glucose concentration; however, glycerol-3P synthesis was higher in the second day of incubation, being largely incorporated into the glycerides-glycerol fraction. Glycerophosphatase expression was not affected by incubation. The stimulation of glycerogenic enzymes’ expression was mirrored in lipases. The result was a shift from medium glycolytic to lipolytic glycerol released as a consequence of increased triacylglycerol turnover, in which most fatty acids were recycled. Production of glycerol seems to be an important primary function of adipocytes, maintained both by glycerogenesis and acyl-glycerol turnover. Production of 3C fragments may also contribute to convert excess glucose into smaller, more readily usable, 3C metabolites.

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.

BackgroundWhite adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown.Experimental DesignEpididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated.ResultsCell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70–75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT.ConclusionsThe methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the “live cell mass” of adipose tissue is very small: about 13 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood (the rats were killed by exsanguination). These data translate (with respect to the actual “live cytoplasm” size) into an extremely high metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

Background. White adipose tissue (WAT) is a complex, disperse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defence, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT breaking up the matrix collagen fibres, but primary cultures of these cells could not be easily correlated to intact WAT, since often recovery and viability are unknown. Experimental design. Epididymal WAT of 4-6 young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of lactate production. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were also measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70-75%. Adipocytes were 7%, erythrocytes 68% and other stromal (nucleated cells) 24% of total WAT cells. However, their overall volumes were, 91%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 2.5% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have found, also, that the "live cell mass" of adipose tissue is very small (about 25 µL/g for adipocytes and 2 µL/g stromal, plus about 1 µL/g blood). This fact, translates into an extremely high (with respect to the actual "live cytoplasm" size) metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

White adipose tissue (WAT) nucleated stromal cells (NSC) play important roles in regulation, defense, regeneration and metabolic control. In WAT sites, the proportions and functions of NSC change under diverse physiological or pathologic conditions. We had previously observed the massive anaerobic wasting of glucose to lactate and glycerol in rat epididymal adipocytes. To test site variability, and whether the adipocyte extensive anaerobic metabolism of glucose was found in NSC, we analyzed, in parallel, subcutaneous, mesenteric and epididymal WAT of male adult Wistar rats. Adipocytes and NSC fractions, were isolated, counted and incubated (as well as red blood cells: RBC) with glucose, and their ability to use glucose and produce lactate, glycerol, and free fatty acids was measured. Results were computed taking into account the number of cells present in WAT samples. Cell numbers were found in proportions close to 1:13:100 (respectively, for adipocytes, NSC and RBC) but their volumes followed a reversed pattern: 7,500:10:1. When counting only non-fat cell volumes, the ratios changed dramatically to 100:10:1. RBC contribution to lactate production was practically insignificant. In most samples, NSC produced more lactate than adipocytes did, but only adipocytes secreted glycerol (and fatty acids in smaller amounts). Glucose consumption was also highest in NSC, especially in mesenteric WAT. The heterogeneous NSC showed a practically anaerobic metabolism (like that already observed in adipocytes). Thus, NSC quantitative production of lactate markedly contributed (i.e. more than adipocytes) to WAT global use (wasting) of glucose. We also confirmed that glucose-derived glycerol is exclusively produced by adipocytes.

BackgroundAdult rat epididymal adipocytes are able to convert large amounts of glucose to lactate and glycerol. However, fatty acid efflux is much lower than that expected from glycerol levels if they were the product of lipolysis. Use of glucose for lipogenesis is limited, in contrast with the active glycolysis-derived lactate (and other 3-carbon substrates). In this study, we analyzed whether white adipose tissue (WAT) site and sex affect these processes.MethodsMature adipocytes from perigonadal, mesenteric and subcutaneous WAT of female and male rats were isolated, and incubated with 7 or 14 mM glucose during 1 or 2 days. Glucose consumption, metabolite efflux and gene expression of glycolytic and lipogenesis-related genes were measured.ResultsThe effects of medium initial glucose concentration were minimal on most parameters studied. Sex-induced differences that were more extensive; however, the most marked, distinct, effects between WAT sites, were dependent on the time of incubation. In general, the production of lactate was maintained during the incubation, but glycerol release rates increased with time, shifting from a largely glycolytic origin to its triacylglycerol (TAG) lipolytic release. Glycerol incorporation was concurrent with increased TAG turnover: lipolytic glycerol was selectively secreted, while most fatty acids were recycled again into TAG. Fatty acid efflux increased with incubation, but was, nevertheless, minimal compared with that of glycerol. Production of lactate and glycerol from glucose were maximal in mesenteric WAT.DiscussionFemale rats showed a higher adipocyte metabolic activity than males. In mesenteric WAT, gene expression (and substrate efflux) data suggested that adipocyte oxidation of pyruvate to acetyl-CoA was higher in females than in males, with enhanced return of oxaloacetate to the cytoplasm for its final conversion to lactate. WAT site differences showed marked tissue specialization-related differences. Use of glucose for lipogenesis was seriously hampered over time, when TAG turnover-related lipolysis was activated. We postulate that these mechanisms may help decrease glycaemia and fat storage, producing, instead, a higher availability of less-regulated 3-carbon substrates, used for energy elsewhere.

White adipose tissue can metabolize large amounts of glucose to glycerol and lactate. We quantitatively traced glucose label to lactate, glycerol and fats in primary cultures of mature rat epididymal adipocytes. Cells were incubated with 7/14 mM C-glucose for 24/48 h. Medium metabolites and the label in them and in cells' components were measured. Gene expression analysis was done using parallel incubations. Glucose concentration did not affect lactate efflux and most parameters. Glycerol efflux increased after 24 h, coinciding with arrested lipogenesis. Steady production of lactate was maintained in parallel to glycerogenesis. Changes in adipocyte metabolism were paralleled by gene expression. Glucose use for lipogenesis was minimal, and stopped (24 h-onwards) when glycerol efflux increased because of triacylglycerol turnover. Lactate steady efflux showed that anaerobic glycolysis was the main adipocyte source of energy. We can assume that adipose tissue may play a quantitatively significant effect on glycaemia, returning 3C fragments thus minimizing lipogenesis.

Under normoxic conditions, adipocytes in primary culture convert huge amounts of glucose to lactate and glycerol. This “wasting” of glucose may help to diminish hyperglycemia. Given the importance of insulin in the metabolism, we have studied how it affects adipocyte response to varying glucose levels, and whether the high basal conversion of glucose to 3-carbon fragments is affected by insulin. Rat fat cells were incubated for 24 h in the presence or absence of 175 nM insulin and 3.5, 7, or 14 mM glucose; half of the wells contained 14C-glucose. We analyzed glucose label fate, medium metabolites, and the expression of key genes controlling glucose and lipid metabolism. Insulin increased both glucose uptake and the flow of carbon through glycolysis and lipogenesis. Lactate excretion was related to medium glucose levels, which agrees with the purported role of disposing excess (circulating) glucose. When medium glucose was low, most basal glycerol came from lipolysis, but when glucose was high, release of glycerol via breakup of glycerol-3P was predominant. Although insulin promotes lipogenesis, it also limited the synthesis of glycerol-3P from glucose and its incorporation into acyl-glycerols. We assume that this is a mechanism of adipose tissue defense to avoid crippling fat accumulation which has not yet been described.