Dihydrocytochalasin B promotes adipose conversion of 3T3 cells

Differentiation of preadipose 3T3-F442A cells into adipose cells is accelerated by the addition of dihydrocytochalasin B. The effect of the drug on 3T3-C2 cells is more marked: these cells are practically unable to differentiate in the absence of H2CB but a long-term exposure to the drug enables the cells to accumulate lipid droplets in medium supplemented with fetal calf serum and insulin. During their differentiation under these conditions the 3T3-C2 cells develop markers typical of adipose cells: glycerophosphate dehydrogenase, ATP-citrate lyase, fatty acid synthetase and glycerophosphate acyltransferase.     

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.     

Gene expression regulation of adipocyte differentiation: cell cycle and hormones

The adipose conversion of cultured preadipose cells involves the activation of numerous genes and is controlled by various adipogenic and mitogenic factors. The differentiation program can be divided into early and late events. Early events are triggered by growth arrest at the G1/S boundary and characterized by the activation of a set of genes (pOb24, lipoprotein lipase, etc.). The expression of the terminal differentiation-related genes takes place after a limited growth resumption of early markers containing cells and requires the presence of permissive hormones (growth hormone and triiodothyronine). Insulin acts solely as a modulator in the expression of the terminal differentiation-related genes. In vivo studies suggest that the acquisition of new adipocytes might result from terminal differentiation of dormant, already committed (pOb24 positive) cells when exposed to appropriate mitogenic or adipogenic stimuli.     

Various stimulators of the cyclic AMP pathway fail to promote adipose conversion of porcine preadipocytes in primary culture

The cyclic adenosine-monophosphate (cAMP) pathway is generally recognized as one of the essential pathways for the adipose conversion of rodent preadipocytes in vitro. However, divergent effects of cAMP on adipocyte differentiation have also been reported. Since there is very little data on non-rodent preadipose cells, the aim of the present work was to analyze the effects of classic activators of the cAMP pathway on the proliferation and differentiation of porcine preadipocytes grown either in serum-free or in serum-containing medium. In both media, the addition of 10 microM forskolin from day 1 after cell plating to day 3 or 7 did not affect cell proliferation. Such stimulations also failed to enhance preadipocyte differentiation, as assessed by the measurement of lipoprotein lipase (LPL) and glycerol 3-phosphate dehydrogenase (GPDH) activities, two markers of adipose conversion. Similar results were obtained when various concentrations of forskolin (0.1 nM-100 microM) were added for 2 days either during the growth phase (days 1-3) or after confluence (days 5-7). Addition of methylisobutylxanthine (MIX) or 8-bromo-cAMP was also found inefficient to stimulate porcine preadipocytes differentiation clearly. By contrast, post-confluence treatment of the murine 3T3-L1 cell line with either forskolin or MIX markedly enhanced lipid accumulation and led to a dramatic increase in GPDH activity (up to 120 times). This indicates that similar culture conditions are adipogenic for the murine 3T3-L1 preadipocytes but not for porcine preadipose cells. In summary, this work clearly highlights the finding that porcine preadipocytes do not respond to classic activators of the cAMP pathway like rodent cells do. This calls in question again the general model proposed for the action of this pathway in adipose conversion and suggests that the mechanisms regulating adipocyte differentiation may differ among species.     

Properties of growth hormone receptors in relation to the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells undergo a process of differentiation in which they convert to adipose cells. Growth hormone promotes this conversion. Since 3T3 sublines vary in their susceptibility to adipose conversion, it was of interest to examine the properties of the growth hormone receptors in relation to that susceptibility. It was found that preadipose 3T3-F442A cells, which are able to convert to adipose cells with high frequency, are able to bind about 10(4) growth hormone molecules per cell with Kd approximately 10(-9) M. After adipose conversion, no appreciable change in hormone binding was detected. The binding of growth hormone to 3T3-C2 cells (a line virtually insusceptible to adipose conversion) was indistinguishable from that to 3T3-F442A cells. Internalization and degradation of the hormone were also similar in the two cell lines. Susceptibility to adipose conversion is therefore not determined by the relative ability of the cells to bind or degrade the hormone, but must instead depend on some response, as yet unidentified, that follows binding of the hormone.     

Alterations of peroxisome proliferator-activated receptor delta activity affect fatty acid-controlled adipose differentiation

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARgamma or PPARdelta promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARdelta in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARdelta mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARdelta enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARgamma and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARdelta mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARdelta plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARdelta expression or activity could affect adaptive responses of white adipose tissue to nutritional changes.     

Control of terminal differentiation of adipose precursor cells by glucocorticoids.

The role of glucocorticoids on adipose conversion has been studied using confluent Ob1771 mouse preadipose cells maintained in a serum-free culture medium able to support the emergence of early but not that of late markers of differentiation. Under these culture conditions, glucocorticoids play, at physiological concentrations, a permissive role for terminal differentiation, characterized by glycerol-3-phosphate dehydrogenase expression and triacylglycerol accumulation within 12 days, whereas progesterone, testosterone, and estradiol are inactive. Glucocorticoids behave as mitogenic-adipogenic stimuli able to trigger growth-arrested, early marker-expressing cells to enter the terminal phase of the differentiation program and thus appear to mimic the mitogenic-adipogenic activity already described for arachidonic acid and cyclic AMP-elevating agents, especially prostacyclin. When compared to corticosterone alone, exposure of Ob1771 cells to both corticosterone and arachidonic acid leads to an additional increase in the glycerol-3-phosphate dehydrogenase activity and number of differentiated cells; this potentiation is further enhanced when the culture medium is supplemented with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. This suggests indirectly the involvement of prostacyclin as a metabolite of arachidonic acid able to induce cyclic AMP accumulation. In agreement with this hypothesis, it is found that a promoting effect is exerted by corticosterone on the metabolism of arachidonic acid, leading in turn to an increase in the production of prostacyclin. These findings allow a better understanding of the role of glucocorticoids on adipose cell differentiation and explain a posteriori the effectiveness of the combination of dexamethasone-isobutyl-methylxanthine used in innumerable studies.     

Specificity of gene expression in adipocytes.

During the differentiation of preadipose 3T3 cells into adipose cells, the mRNAs for three proteins increase strikingly in abundance. To determine the degree of cell-type specificity in the expression of these mRNAs, we estimated their abundances in several nonadipose tissues of the mouse. None of these mRNAs was strictly confined to adipocytes, but the ensemble of three mRNAs was rather specific to adipocytes. Insofar as is revealed by these three markers, the distinctive phenotype of adipocytes is the result of the enhanced expression of a number of genes, none of which is completely silent in all other cell types.     

Preadipocyte differentiation in vitro: identification of a highly active adipogenic agent

A highly active adipogenic agent was identified in an in vitro adipose conversion system. This agent, ADD 4743 (or ADD), was synthesized by Takeda Chemical Industries (Osaka) as a 3-hydroxy derivative of an oral antidiabetic agent, ciglitazone, and has been presumed to be an active metabolite of the latter substance. When ST 13 mouse preadipose cells were treated with micromolar concentrations of ADD they rapidly and uniformly converted into lipid-accumulating adipocytelike cells within 8-11 days after cell seeding. The degree of adipose conversion and lipid accumulation induced by ADD far exceeded those of the previously known inducing agents such as indomethacin plus insulin. The highly potent adipogenic activity of ADD was confirmed with two other preadipose cell lines (3T3 L1 and RMT rat preadipose cells). In addition to adipogenic activity, ADD inhibited cell proliferation of preadipose cells specifically. Activity of ADD induced lipid accumulation and growth inhibition of ST 13 cells, exhibiting very similar dose-response relationships. Cell proliferation or triacylglycerol content of nonadipocytic mesenchymal cells or epithelial cells were not affected by ADD. These observations strongly suggest that ADD-induced growth inhibition is not due to the nonspecific toxicity of the drug but is tightly associated with the adipocytic character of the treated cells. The present observation provides evidence that ADD would be a powerful agent in studies that involve preadipocyte differentiation.     

Fetuin modulates growth and differentiation of Ob17 preadipose cells in serum-free hormone-supplemented medium

A serum-free hormone-supplemented medium able to support the growth of rodent adipose precursor cells has been used to characterize additional components from serum required for the differentiation of preadipose Ob17 cells into adipose-like cells. Fetuin is shown to behave as a growth-promoting agent for these cells. In addition to growth hormone, triiodothyronine and a low-molecular weight component(s) also purified from serum, fetuin is required for the full expression of the differentiation program. Other serum proteins as well as other mitogenic factors are unable to substitute for fetuin. A possible role of fetuin in the development of adipose tissue is discussed.     

Cloning and regulation of a mRNA specifically expressed in the preadipose state

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.     

Activity of human growth hormone and related polypeptides on the adipose conversion of 3T3 cells.

A culture system is described for the study of cellular responsiveness to growth hormone. The hormone acts directly on an established line of preadipose 3T3 cells and promotes their differentiation into adipose cells. This response is the basis of a sensitive and specific assay and does not depend on the participation of an intermediate effector.     

Inhibition of adipose conversion of BALB/c 3T3 cells by interferon and 12-O-tetradecanoylphorbol-13-acetate

The adipose conversion of BALB/c 3T3 preadipose cells is inhibited by interferon; this inhibition is directly correlation with the interferon concentration. In cultures treated with low doses of interferon and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, another inhibitor of adipose conversion (Diamond et al., 1977), the two compounds act synergistically to block differentiation. Several lines of evidence suggest that the compounds differ in the mechanism by which they inhibit adipose conversion.     

The prevention of adipose differentiation of 3T3-L1 cells caused by retinoic acid is elicited through retinoic acid receptor alpha

Retinoids, especially all-trans retinoic acid (RA), have been shown to inhibit the differentiation of preadipose cells. It is important to human health, especially to obesity, that the regulatory system for the differentiation of adipocytes is well defined. Previously, we have shown that retinoic acid receptor (RAR) γ2 gene expression is up-regulated by RA in 3T3-L1 preadipose cells. In this study, the RAR system was dissected and the RA-regulated function in 3T3-L1 cells was assigned to one given receptor. We used three synthetic retinoids; (1) Ro 41–5253, a selective RAR α antagonist, (2) Ch 55, an RAR α, β and γ agonist, and (3) Am 80, an RAR α and β agonist, which has less affinity to RAR γ. Ro 41–5253 reverted RA-induced inhibition of the differentiation of 3T3-L1 cells. However, there was no significant reversion in RA-induced RAR γ mRNA level by treatment with Ro 41–5253. In the case of RAR agonists, both Am 80 and Ch 55 strongly inhibited the differentiation of 3T3-L1 cells. However, Am 80 weakly increased RAR γ mRNA content less than did Ch 55. These findings suggest, that RAR α is involved in the prevention of adipose differentiation by RA in 3T3-L1 cells. Moreover, there seems no causal relationship between the prevention of adipose differentiation by RA and the up-regulation of RAR γ2 gene expression by RA in 3T3-L1 cells. We have shown the functional heterogeneity of RA action through different RARs in 3T3-L1 cells.     

Extracellular matrix induced by TGFβ impairs insulin signal transduction in 3T3-L1 preadipose cells

When 3T3-L1 preadipose cells are exposed to transforming growth factor β (TGFβ), they synthesize more extracellular matrix (ECM) and resist differentiation-inducing stimuli. The mechanism by which ECM suppresses adipose cell differentiation (adipogenesis) remains unknown. Since adipogenesis is an insulin/insulin-like growth factor-1 (IGF-1)-dependent process, we investigated whether TGFβ-induced ECM inhibits insulin signaling. When preadipose cells were pretreated overnight with TGFβ, we observed a 75% decrease in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) compared to that in control cells. Culturing 3T3-L1 preadipose cells on fibronectin, a component of the ECM induced by TGFβ, also inhibited insulin-dependent IRS-1 tyrosine phosphorylation and adipogenesis, supporting a role for ECM in mediating TGFβ's inhibitory effect on insulin signaling. Since the insulin-stimulated association of phosphoinositide (PI) 3-kinase with IRS-1 depends on IRS-1 tyrosine phosphorylation, we measured the presence of the PI 3-kinase 85 kDa regulatory subunit in anti-IRS-1 immunoprecipitates. Following insulin stimulation, PI 3-kinase-IRS-1 association was reduced by 70% in TGFβ pretreated vs. control preadipose cells. However, insulin-stimulated cellular production of PI(3,4,5)P3 was unaltered by TGFβ pretreatment. This suggests that IRS-1-associated p85-type PI 3-kinase may represent a particular subset of total cellular PI 3-kinase that is specifically inhibited by TGFβ. Reduction of insulin-stimulated association of IRS-1 with p85-type PI 3-kinase by TGFβ may be one potential mechanism through which TGFβ blocks 3T3-L1 adipose cell differentiation. J. Cell. Physiol. 175:370–378, 1998. © 1998 Wiley-Liss, Inc.     

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

Background  

Multipotent 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.     

Prostaglandin F2 alpha inhibits the differentiation of adipocyte precursors in primary culture.

Influence of arachidonate metabolite pathway on adipose differentiation was investigated using primary culture of adipocyte precursors in defined medium. Treatment of the cells with cyclooxygenase inhibitors stimulates adipose differentiation by at least 2-fold. Among the various arachidonate metabolites tested, only prostaglandin F2 alpha (PGF2 alpha) was found to inhibit the differentiation of adipocyte precursors in a dose dependent fashion. Other eicosanoids tested did not have any effect. A 50% inhibition of adipose differentiation was observed with a dose of PGF2 alpha of 3 x 10(-9)M to 7 x 10(-9)M according to the strain of rats used. Maximal inhibition occurred at PGF2 alpha concentrations equal or higher than 10(-8)M. PGF2 alpha inhibited not only the expression of late markers of adipose differentiation such as G3PDH and triglycerides accumulation but also the mRNA expression of early markers of adipose differentiation such as clone 154, lipoprotein lipase and ap2 gene. These results indicate that PGF2 alpha represents a physiological negative modulator of adipose differentiation.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

Differentiation-associated increase of cAMP-dependent type II protein kinase in a murine preadipose cell line (ST 13)

The activity of cAMP-dependent protein kinase and cAMP binding activity were studied during the differentiation of ST 13 murine preadipocytes into adipocytes. We found that both activities were marginally detectable in preadipose cells and increased remarkably when the cells were induced to differentiate, preceding by several days the morphological adipose conversion. The increased cAMP-dependent protein kinase was identified as type II enzyme by means of DEAE-Sephacel chromatography and by photoaffinity labeling with 8-azido[3H]cAMP. We further showed that the increase of protein kinase activity was specific to cell differentiation with the aid of modulators of the adipose conversion (insulin, fetal bovine serum, retinoic acid and 5-bromodeoxy-uridine). We propose that the increased expression of type II cAMP-dependent protein kinase would be a biochemical index of differentiation in ST 13 preadipocytes.     

A method for separating cultured preadipocytes according to their density: Application to stromal cells from overfed suckling rats

Summary The stroma vascular fraction of adipose tissue consists of a heterogeneous cell population; not all the cells in this compartment undergo adipose conversion in primary culture. A density gradient centrifugation procedure was used to separate cultured cells on the basis of their triglyceride content. This method was applied to both stroma vascular cells from rat adipose tissue and to a 3T3 F442A preadipose cell line as a reference. Comparison of the results obtained from these two cell types suggests that this separation procedure can lead to a quantification of adipose differentiation in the heterogeneous stroma cell population. Separation procedures were applied to cultured stromal cells derived from young rats during the onset of nutritional obesity induced by overfeeding in early life. Results show that early overfeeding induced an increase in the stromal cell differentiation capacity which is expressed in vitro. This work was supported in part by Institut National de la Santé et de la Recherche Médicale (CRL n^o 82-70-22).