Quantitative analysis of rat adipose tissue cell recovery, and non-fat cell volume, in primary cell cultures

Abstract

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.

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

“…The metabolic prowess of WAT, a tissue with so small proportion of 'live' cytoplasm [25], does not cease to surprise us with a widespread and powerful participation in the overall control of body energy . Also by the growing number of functional metabolic pathways it contains and 'hides' in between so much fat.…”

“…Adipocytes were isolated by incubation with collagenase as described in a previous paper [25], essentially following the Rodbell procedure [48]. Cells were counted, and their (spherical) diameters measured using the ImageJ software (http://imagej.nih.gov/ij/) [49].…”

“…Cells were counted, and their (spherical) diameters measured using the ImageJ software (http://imagej.nih.gov/ij/) [49]. The yield with respect to WAT sample mass was estimated in a number of randomly selected samples as previously described [25]. The adipocytes recovered were in the range of 73-75 % of those present in the tissue.…”

“…The conversion of glucose into 3C fragments, together with the low lipogenic activity from glucose and high recycling rate of lipolytic-freed fatty acids shown by incubated adipocytes [12,24] has been attributed, largely, to the geometry of mature (large) adipocytes. 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].…”

“…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.…”