Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone.

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.

Development of insulin and epidermal growth factor receptors during the differentiation of rat preadipocytes in primary culture.

An homogeneous cell population isolated from the inguinal tissue of 3-day-old rats is able to proliferate in primary culture. In the presence of a physiological concentration of insulin (1.5 nM) it converts into cells exhibiting the morphology and the biochemical characteristics of adipocytes. Insulin and epidermal growth factor (EGF) receptors were studied during both the exponential growth and the adipose conversion phases of these cells. Binding experiments with 125I-labelled peptides were performed directly in the culture dishes. The number of high affinity insulin binding sites increased, during the entire culture period studied, reaching 18 days after plating the value of 10,600 x 2360. Control cells (cultured in the presence of anti-insulin antibody) exhibited an increase of the concentration of insulin binding sites from no more than 500 sites/cell to 6880 +/- 1710 sites/cell between dat 0 and 9 (corresponding to the exponential growth phase); this increase was followed by a rapid reduction in insulin receptors during the stationary phase. The density of EGF binding sites increased between day 0 and 4 (one cell cycle), whether the cells were maintained or not with insulin, and plateaued thereafter. Mature adipocytes freshly isolated from the inguinal tissue of 3-day-old rats had no detectable EGF binding sites, but their content in high affinity binding sites for insulin was similar to that of cells after complete adipocyte conversion in primary culture.

[Characterization and differentiation of rat preadipocytes in primary culture]. / Caractérisation et différenciation de préadipocytes de rats en culture primaire.

Using a density gradient medium, we isolated homogeneous cell populations from the inguinal tissue of 3-day old rats. In primary culture we obtained, for the first time, the differentiation of 99% of the cells in the presence of a physiological concentration of insulin (10(-9) M). This model closely mimicked the events occurring during normal mammalian adipose development, i.e. a positive change in beta-adrenergic sensitivity, early induction of lipoprotein lipase, expression of the enzymes involved in the triglyceride systems, and the development of responsiveness to glucagon.

Characteristics of the rat adipoblast during differentiation in culture.

In the present study, stromal vascular cells were obtained from the inguinal fat pad of 3-day-old rats and layered on a continuous density gradient (Percoll). After centrifugation, it was possible to collect three homogeneous cell populations of different densities and to grow them separately in primary culture. Upon reaching a confluent state two of them particularly (the lighter fractions) converted to adipocytes with a high frequency (90 per cent) in the presence of physiological concentration of insulin (10(-8) M). During the adipose conversion insulin markedly enhanced the activities of lipogenic enzymes. When VLDL (350 micrograms lipids/ml) and heparin (10 IU/ml) were added to the medium, this effect was not potentiated. However VLDL and heparin in presence of insulin increased the triacylglycerol content of the differentiated cells. The heavier fraction did not undergo adipose conversion to the same extent as the lighter ones. It was concluded that the high ability of these precursor cells to convert to adipocytes and their response to low concentration of insulin could be related to the early development stage of the tissue.

Development under the control of insulin of lipogenic enzymes, lipoprotein lipase, isoproterenol and glucagon sensitivity in differentiating rat preadipocytes in primary culture.

In preadipose cellular fractions (I, II and III) isolated by density gradient centrifugation from the inguinal tissue of young rats, we followed the activity of fatty acid synthetase, ATP citrate lyase and lipoprotein lipase during differentiation in culture. 1.5 nM insulin when added at confluence markedly induced the activity of ATP citrate lyase and fatty acid synthetase in the cells derived from the lighter fractions (I and II). The magnitude of this response was 25-50-fold the initial value 15 days after plating. In the cells of the heaviest fraction (III) both enzymes exhibited low activity which was slightly stimulated by the presence of insulin, VLDL and heparin. In contrast, the activity of lipoprotein lipase appeared before confluence in cells from all three fractions and peaked at day 6 after plating. This early emergence was independent of the addition of insulin to the medium. However, insulin slightly enhanced the peak activity in post-confluent cells. The development of cAMP production in response to isoproterenol (100 microM) and to glucagon (0.3 microM) was determined in the cells of fraction II in the same culture conditions. The responsiveness to isoproterenol was present very early in these cells and rose rapidly during the exponential growth phase, reaching a peak value at day 8 after plating. In contrast, the development of glucagon sensitivity occurred only during late differentiation. The stimulatory effect of glucagon was enhanced when VLDL and heparin were added with insulin to the medium.

Differentiation under the control of insulin of rat preadipocytes in primary culture. Isolation of homogeneous cellular fractions by gradient centrifugation.

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10(-9) M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid: CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.

Alteration, by early underfeeding, of cellular multiplication and differentiation in the inguinal fat pads of rats.

The effect of litter size on the incorporation of labeled thymidine (TdR) into DNA was studied in the stromal and the adipocyte fractions of the rat inguinal tissue. In experiment 1 the animal were kept in litters of 18 (UF) or 6 (control) from birth till 10 days. They were injected with [2-14C] TdR at day 3 and killed at 60 minutes, 1, 3 and 7 days post-injection. In experiment 2, the pups were raised in litters of 18 during 3 (RF3), 6 (RF6) or 10 (RF10) days, and distributed again in litters of six. They were injected with [2-14C] TdR or [14CH3]TdR at the beginning of the refeeding and killed as described previously. In all experiments the weight of the inguinal tissue was more reduced than the total body weight. In the UF, the proliferation was markedly reduced in cellular fractions as was the differentiation of stromal cells into adipocytes from six days of underfeeding. In the RF3 and the RF6 there was an attempt to recover the cell number as suggested by the recycling of the degradation products of TdR for DNA synthesis. In the RF10, cell multiplication and differentiation were strongly affected by the length of the deprivation period.

Studies on cell proliferation in inguinal adipose tissue during early development in the rat.

[2-14C] Thymidine was injected into rats aged 3,5 and 10 days, and incorporation of the precursor into deoxyribonucleic acid (DNA) of the inguinal fat tissue was measured for short time periods. Using chromatographic procedures to measure the distribution of thymidine and its metabolites in the soluble fraction of the tissue, degradation of the precursor was found to be similar at all ages. The data indicate that thymidine was more rapidly utilized for DNA synthesis in 3-day-old rats than in older animals. When 14C-thymidine was injected in vivo and adipocytes and stromal cells were then separated from the inguinal tissue of 3-and 5-day-old rats, the incorporation into DNA was significant in both types of cells already 30 min after pulse labeling. Stromal cells took up twice as much of label as the adipocytes. Furthermore, real incorporation into DNA was found in the adipocytes when incubated in vitro in a culture medium supplemented with 14C-thymidine. The possibility is discussed that early in postnatal life adipocytes might synthesize DNA for further cell division.