Differentiation Of Preadipose Cells Research Articles

Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

BackgroundMultipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.ResultsOur results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.ConclusionThese results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

Differentiation of preadipose cells: paracrine role of prostacyclin upon stimulation of adipose cells by angiotensin-II

Differentiation of preadipose cells: paracrine role of prostacyclin upon stimulation of adipose cells by angiotensin-II

Differentiation of preadipose cells: paracrine role of prostacyclin upon stimulation of adipose cells by angiotensin-II.

Prostacyclin (PGI2), the major metabolite of arachidonic acid in adipose tissue, has been shown to play a key role in the process of preadipose cell differentiation in vitro. Moreover, angiotensin-II (Ang II) is able to induce the production of PGI2 in suspensions of isolated adipocytes as well as in the interstitial fluid of rat adipose tissue. A possible role of Ang II in the control of the autocrine-paracrine adipogenic effect of PGI2 has been investigated, using cells of the Ob1771 preadipocyte clonal line cultured in serum-free chemically defined medium. Whereas both preadipose and adipose cells were able to produce PGI2 upon exposure to arachidonic acid, only adipose cells were able to do so when challenged with Ang II. In agreement with this observation, the ability of Ang II to induce preadipose cells to differentiate required the simultaneous presence of differentiated cells. Such coculture experiments show that the promoting effect of Ang II on preadipose cell differentiation was strongly reduced by aspirin, antibodies able to neutralize PGI2, and the AT2 receptor antagonist PD123177, but not by the AT1 receptor antagonist losartan. Together, these results support Ang II as being able, by means of binding to a receptor of the AT2 subtype present in adipose cells, to control the adipogenic effect of PGI2 through a paracrine mode of action.

Essential role of collagens for terminal differentiation of preadipocytes

In order to study the role of collagens in the differentiation of TA1 preadipose cells in vitro , ethyl-3,4-dihydroxybenzoate (EDHB) was used as a specific inhibitor of collagen synthesis. The secretion of collagenous proteins only was severely decreased after exposure to EDHB, and this was accompanied by a decrease of differentiation as indicated by low activity levels of glycerophosphate dehydrogenase. The effect of EDHB was dose-dependent and also dependent upon the stage of cell differentiation. Northern-blot analysis show that EDHB addition to undifferentiated cells did not prevent the induction of A2COL6 gene, a marker of the preadipose state, but prevented the induction of the gene encoding for the adipocyte lipid binding protein and the modulation of the expression of the lipoprotein lipase gene which are both indicators of the adipose state. These results demonstrate that differentiation of preadipose cells into adipose cells requires active synthesis of collagens during the preadipose state.

Requirement and role of arachidonic acid in the differentiation of pre-adipose cells

The terminal adipose differentiation of Ob1771 cells, characterized by glycerol-3-phosphate dehydrogenase activity and triacylglycerol accumulation, was studied in serum-free hormone-supplemented medium containing growth hormone, tri-iodothyronine, insulin, transferrin and fetuin. Arachidonic acid was able to substitute for a crude adipogenic fraction isolated from fetal bovine serum but not for growth hormone or tri-iodothyronine. Arachidonic acid was also able to increase in a rapid and dramatic manner cyclic AMP production; moreover it was able to amplify the adipose conversion promoted by other agents elevating cyclic AMP concentrations and to induce inositol phospholipid breakdown. Both phorbol 12-myristate 13-acetate, a protein kinase C activator and ionomycin, a Ca2+-mobilizing agent, showed potent synergy with agents elevating cyclic AMP concentrations for the promotion of adipose conversion, whereas 8-bromo cyclic GMP and 4 alpha-phorbol 12,13-dibutyrate were ineffective. The triggering of both the cyclic AMP and inositol phospholipid pathways was accompanied by a single round of cell division, and within a few days all the cells became differentiated. Similar results were obtained, after exposure to arachidonic acid, with preadipose 3T3-F442A cells and with rat adipose precursor cells in primary culture. The availability of arachidonic acid from intracellular stores and/or of exogenous origin should play a major role for the onset of critical mitoses leading to terminal differentiation in pre-adipose cells.

Suppression of SV40-promoted gene expression by differentiation of preadipose cells.

When a plasmid bearing the chloramphenicol acetyltransferase (CAT) gene under the control of an SV40 early promoter is introduced into preadipose or adipose cells of line 3T3-F442A, the promoter directs high levels of transient expression of CAT. However, when the plasmid is introduced into preadipose cells and the cells are then allowed to differentiate into adipose cells, the expression of the CAT gene is suppressed. In this process, the plasmid is not changed detectably in amount, topology, or state of methylation. Stably transformed preadipose cells bearing an integrated plasmid express the transferase, but if the cells are allowed to differentiate, the expression of the gene is similarly suppressed. The decline in CAT activity is associated with a decrease in the transcription rate of the gene. Transcription of a gene coding for neomycin phosphotransferase driven by the SV40 promoter is also greatly diminished by differentiation. Because suppression of CAT does not occur when the gene is under control of a retroviral long terminal repeat (LTR), a specific mechanism exists for the recognition and inactivation of the SV40 early promoter during differentiation.