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.     

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.     

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.     

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.     

Growth hormone and the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells are induced or enabled to undergo adipose conversion in the presence of an extract of pituitary gland. Adipogenic activity is found in standard growth hormone preparations derived from different species. Further purification of rat growth hormone by several methods does not remove its adipogenic activity. Human growth hormone synthesized in Escherichia coli is also effective. Adipogenic activity is not associated with other pituitary polypeptides. Since growth hormone acts on preadipose cells in the absence of any other cell type, a mechanism exists for the direct participation of the pituitary gland in the regulation of this form of mesenchymal differentiation.     

Growth arrest by octanoate is required for porcine preadipocyte differentiation

A preadipocyte clonal line has been established from porcine subcutaneous tissue. This line, designated PSPA, showed a fibroblastic phenotype and kept on growing under a preadipose condition even after reaching confluence. When confluent cultures were stimulated with insulin, dexamethasone, biotin, pantothenate, and octanoate, growth was arrested, and the cells exhibited a marked increase in lipogenesis. However, adipose conversion was not induced upon exposure of PSPA cells to a standard hormonal mixture of mouse 3T3-L1 cells, and they continued dividing as did the preadipocytes in growth medium. By serially omitting each individual adipogenic agent from the PSPA differentiation medium, it was determined that octanoate was one of the most essential but the only factor able to induce growth arrest. Octanoate supplementation to 3T3-L1 medium increased the triglyceride accumulation of PSPA cells accompanied by growth arrest. Both RT-PCR and Western blot analysis supported the idea of octanoate as a potential agent with the antiproliferative activity requisite for porcine preadipocytes to enter terminal differentiation.     

Prostacyclin as a critical prostanoid in adipogenesis

Adipose cell differentiation from adipoblasts to preadipose and to adipose cells is a multistep process. Terminal differentiation of preadipose cells expressing early markers to adipose cells expressing late and very late markers and accumulating triacylglycerol requires a combination of circulating and locally-produced hormones. Prostacyclin (PGI2), one of the major metabolites of arachidonic acid in adipose tissue, has been shown to exert autocrine and paracrine adipogenic effects in vitro. As discussed herein, multiple arguments support the proposition that PGI2 is a key prostanoid involved in adipogenesis.     

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.     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Induction of adipose conversion by mouse serum in cultured cells and its characterization

Mouse serum (MS) effected a rapid accumulation of many lipid droplets by cultured cells in the growing or resting state. MS-induced adipose conversion in all of 12 randomly selected cell lines, including human, mink, rat, and mouse cells and almost all of the cells in a culture dish were converted. Under excessive amounts of MS, the cells became mature adipocytes, lost the ability to divide and soon died. However, proliferation of adipocytes induced by smaller quantities of MS was not different from that of control cells in calf serum (CS). When adipose conversion developed, oncorna virus producing cells ceased virus production and there was a clear connection between decrease of virus production and rate of adipose conversion. The adipose conversion of 3T3-FL cells grown for 7 days in the presence of bromodeoxyuridine (BUdR) was inhibited. Actinomycin D (actD) and cycloheximide also inhibited adipose conversion. It is suggested that the cells may have an inherent ability to differentiate into adipocytes.     

Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24-36 h after adipogenic stimulation: TNF-alpha inhibits commitment

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-alpha at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-alpha inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.     

The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line

Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR γ and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC₅₀ at about 1 nM. The induction medium up-regulated expression of PPARγ and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency. This revised version was published online in July 2006 with corrections to the Cover Date.     

High expression of apolipoprotein E impairs lipid storage and promotes cell proliferation in human adipocytes

Apolipoprotein E (apoE), a key regulator of lipid metabolism, is highly produced by adipose tissue and adipocytes. However, there is little information about its role on adipocyte functions. Because apoE‐deficiency in adipocytes was shown to impair adipocyte differentiation, we investigated the consequences of apoE high expression on differentiation and proliferation of a human adipocytic cell line (SW872). SW872 cells were transfected with human apoE to induce a fivefold increase in apoE production and secretion. Adipocyte differentiation and proliferation were assayed by measuring lipid content, adipogenic gene expression, cell number, cell resistance to serum deprivation, and cell division kinetics. Cultured apoE‐transfected cells accumulated less triglycerides and less cholesterol than control cells. This decrease in lipid accumulation was associated with a strong downregulation of peroxisome proliferator‐activated receptors γ1 and γ2 and stearoyl‐CoA desaturase 1. The decrease in lipid accumulation was not dependent on the presence of lipids, lipoproteins, or PPAR‐γ agonists in the culture medium, nor was it observed with exogenously added apoE. Moreover, we observed that apoE‐transfected cells were more resistant to death induced by serum deprivation, and that these cells underwent more cell divisions than control cells. These results bring new evidence of apoE‐involvement in metabolic disorders at the adipocyte level. J. Cell. Biochem. 106: 608–617, 2009. © 2009 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.     

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate stimulates lactate production in BALB/c 3T3 preadipose cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which reversibly inhibits the adipose conversion of $\text{BALB}\text{c}$ 3T3 preadipose cells, increases lactate production by these cells. The stimulation of lactate production requires 4–7 days for optimal effect. Once TPA is removed from the cultures, the rate of lactate production falls to control levels. The concentration dependence for the TPA-mediated stimulation of lactate production is similar to that for its inhibitory effect on adipose conversion. Exogenous lactate in the absence of TPA also inhibits adipose conversion. These results suggest that the ability of TPA to interfere with the normal pattern of glucose metabolism may be important in the inhibitory effect of TPA on triglyceride accumulation in these cells.     

An established pre-adipose cell line and its differentiation in culture

The established cloned line, 3T3-L1, is a preadipose line. When the cells enter a resting state, either in monolayers or in suspension culture stabilized with methyl cellulose, they accumulate triglyceride fat and become adipose cells. A high serum concentration in the culture medium increases the rapidity and extent of the fat accumulation. The adipose conversion can be delayed indefinitely in surface cultures by keeping the cells in a growing state.3T3-L1 is also specialized for collagen synthesis; prior to its adipose conversion, it makes about as much collagen as other 3T3 cells. We may therefore regard 3T3-L1 as a fibroblast line with an additional form of specialization.After 3T3-L1 cells are grown to confluence in the presence of low concentrations of bromodeoxyuridine, their rate of collagen synthesis is not affected, but their conversion to adipose cells is completely prevented. If the cells are then permitted to grow in medium free of bromodeoxyuridine, their ability to convert to adipose cells is regained. The conversion of 3T3-L1 from pre-adipose to adipose cells therefore involves a process of differentiation which can be studied under cell culture conditions.     

Commitment of 3T3-F442A cells to adipocyte differentiation takes place during the first 24-36 h after adipogenic stimulation: TNF-α inhibits commitment

We studied the commitment of 3T3-F442A cells during stimulation with adipogenic serum or growth hormone. Confluent 3T3-F442A preadipocytes were incubated with adipogenic medium for increasing times; the number of adipose clusters, GPDH activity, and lipid accumulation were evaluated. Results show that cell commitment took place during the first 24-36 h after stimulation under adipogenic conditions. Then, cultures underwent a 2-fold increase in total cell number through selective multiplication of committed cells, followed by a dramatic decrease in colony-forming ability and 300- to 1000-fold raise in GPDH activity. Cell commitment was not modulated by insulin, but this hormone stimulated clonal expansion of committed cells and lipogenesis. Commitment was inhibited by TNF-α at concentrations as low as 5 ng/ml, and by retinoic acid. The results show that TNF-α inhibits adipose conversion at two different levels: at concentrations as low as 5 ng/ml, it blocks commitment, and at concentrations of 100 ng/ml or higher the cytokine seems to block mitotic expansion and other steps of differentiation after cell commitment. The identification of a specific time for cell commitment would allow the study of the early genes that might regulate cell reprogramming into adipocytes.     

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.