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.

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.

Development of mossy fiber synapses in hippocampal slice culture.

The mossy fiber synaptogenesis has been studied in hippocampal slice cultures. In vivo mossy fiber terminals contact the thorny excrescences of CA3 pyramidal neurons over a restricted portion, i.e. the proximal part of the apical dendrite. In organotypic cultures mossy fibers expand their terminal field and invade the infrapyramidal area of the CA3 region and the supragranular layer of the dentate gyrus. Newly formed mossy fiber synapses in CA3 region were examined, through electron microscopy, in cultures taken at various time intervals. The main events of the formation of newly formed mossy fiber synapses can be summarized as follows. During the first week following explantation mossy fiber axons contact the dendritic shaft of the pyramidal dendrite and establish both symmetrical and asymmetrical contacts. Subsequent modifications occur in the postsynaptic portion facing the mossy fiber bouton: (i) a massive accumulation of polyribosomes and coated vesicles in the subsynaptic cytoplasm; (ii) undulations of the plasma membrane; (iii) disappearance of neurotubules at postsynaptic sites and appearance of a fine network of filamentous material. Later on in culture, complex giant spines invaginate within the synaptic bouton. In conclusion this study shows that CA3 pyramidal neurons following deafferentation retain the capacity to form thorny excrescences, when contacted by mossy fibers. Moreover these results suggest a crucial role for mossy fibers to induce the formation of thorny excrescences in an heterotopic localization, i.e. over the basilar dendrites of CA3 pyramidal neurons.

Ontogeny of glucagon and isoproterenol induced cyclic 3'-5' adenosine monophosphate production in adipocytes isolated from the inguinal adipose tissue of developing rats.

To investigate any differences that might exist between the maturation of receptors coupled to the cyclic-AMP system, the effects of glucagon and isoproterenol (specific beta-adrenergic agonist) on the stimulation of cyclic-AMP production in adipocytes and stromal cells isolated from 3, 10 and 24 days-old rats were studied. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine and at a cell concentration of approximately 5 X 10(5) cells/ml, the cyclic-AMP accumulation produced by 100 microM isoproterenol in the stromal vascular cells represented a two-fold and a four-fold increase above basal at 3 days and 24 days respectively, after 10 min incubation at 37 degrees C. In the same conditions, at a cell concentration of 1.7 X 10(5) cells/ml, the increase of cyclic-AMP concentration was maximum in the mature adipocytes after 10 min incubation and represented a 12-fold increase above basal values at all ages. In the latter cells, a striking difference was observed in the level of cyclic-AMP concentrations, induced by maximal doses of glucagon (0.3 microM), according to age. Indeed, at 24 days, the values of glucagon-stimulated cyclic-AMP concentrations, as well as the ratio of stimulated to basal values, was significantly higher than at 3 or 10 days. After 5 min incubation, this increase was two-fold for 3 and 10 days. After 5 min incubation, this increase was two-fold for 3 and 10 days old and four-fold for 24 days-old animals. These results suggested that the beta-adrenergic receptors develop very early in life since they are efficient even in the stromal cells, whereas a progressive development of the glucagon-induced effect could be observed during the suckling period in the lipid laden cells.

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.

Desoxyribonucleic acid and pyrimidine synthesis in the rat during intra-uterine growth retardation: responsiveness of several organs.

The response of DNA synthesis, thymidine incorporation and the activities of uridine kinase, carbamyl phosphate synthetase and aspartate transcarbamylase were studied in the rat, following intra-uterine growth retardation. A good correlation was found between the falls of DNA production and thymidine incorporation and the decrease of the activity of the three enzymes during the fetal period. After birth, cell proliferation remained depressed but no further changes occurred in the enzymatic activities.

[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.