Preadipocytes In Primary Culture Research Articles

Expression of the mitochondrial uncoupling protein in brown adipocytes: Absence in brown preadipocytes and BFC-1 cells. Modulation by isoproterenol in adipocytes

The expression of the uncoupling protein has been compared in cells of BFC-1 clonal line established from mouse brown adipose tissue (BAT) and in preadipocytes, as well as in adipocytes from mouse BAT, both in primary culture. The results of immunoblots show that, after one week in culture, adipocytes have a reduced level of the 32 kD protein. This level can be raised 2–3.5-fold by a 24-h exposure to isoproterenol. Thus a direct modulation by a β-agonist drug in the expression of the uncoupling protein is observed. Under the same conditions as well as under various other conditions, preadipocytes in primary culture and BFC-1 cells do not express the uncoupling protein. At the same time these cells are able both to differentiate into adipose cells, as demonstrated by the emergence of enzyme markers and triglyceride accumulation, and to respond to isoproterenol. Thus isoproterenol is not sufficient to trigger the expression of the uncoupling protein and behaves as a mere modulator once the cells have acquired the capacity to express it. Injection of undifferentiated BFC-1 cells into athymic mice bearing catecholamine-containing mini-osmotic pumps, or co-cultures of BFC-1 cells and pheochromocytoma PC-12 cells do not allow BFC-1 cells to express the uncoupling protein. Taken together, the results suggest that the formation of brown preadipocytes is critically linked during development to the release by sympathetic nerves of specific trophic factors acting locally.

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 μM) and to glucagon (0.3 μM) 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.

Hormonal responsiveness of a preadipose cell line derived from newborn mouse calvaria.

We established a clonal preadispose cell line from newborn mouse calvaria. Cells of this cell line, designated MC3T3-G2/PA6, had the capacity to convert to adipose cells, to accumulate triglycerides in their cytoplasm, and to mature to differentiated fat cells in a resting state. This adipose conversion was markedly accelerated by addition of dexamethasone, which was the most potent inducer among the steroid hormones tested. The presence of dexamethasone was needed during the steroid hormones tested. The presence of dexamethasone was needed during logarithmic growth phase for maximal conversion. The frequency of adipose conversion was dependent on exposure time to the hormone, but cells already committed to differentiation continued to accumulate lipid and developed into mature adipose cell even in its absence. This indicates that the hormone accelerates the initiation of the adipose conversion, but is not required for the ongoing conversion process. In fact, it was rather inhibitory for the process of fat accumulation. Insulin alone slightly inhibited the adipose conversion, but its combination with dexamethasone neutralized the above inhibitory effect of dexamethasone. The responsiveness of this cell line is consistent with that observed for mouse bone marrow preadipocytes in primary culture but differs from that for preadipose cell lines derived from extramedullary tissues. These results strongly suggest that the MC3T3-G2/PA6 cell line was derived from bone marrow.