Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium.

Stromal-vascular cells obtained from adult human subcutaneous adipose tissue were cultured in a chemically defined serum-free medium. In the presence of 0.2 nM triiodothyronine and 0.5 microM insulin, up to 25% of the cells were able to undergo terminal adipose differentiation within 18 d, as assessed by lipid accumulation and the expression of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities. Addition of cortisol resulted in a potent dose-dependent stimulation of the adipose differentiation process. Cortisol could be replaced by dexamethasone and partly by aldosterone, but not by sex steroids. The proportion of differentiated cells was dependent upon the age of the donor; when isolated from young adults, up to 70% of the stromal-vascular cells expressed the adipocyte phenotype as compared with 5-10% when the cells were isolated from the oldest subjects. An inverse relationship was observed between the age of the 27 normal-weight donors and the extent of GPDH expression after maintenance of the cells for 18 d in chemically defined medium supplemented with insulin, triiodothyronine, and cortisol (r = -0.787, P less than 0.001). It is concluded that adult human adipose tissue still contains precursor cells that are able to undergo adipose differentiation in vitro. This improved culture system may offer the opportunity to characterize other adipogenic factors as well as antiadipogenic factors involved in the control of adipose tissue growth.

Prostacyclin as an indicator of preadipocyte transformation: studies in vivo by microdialysis and in vitro.

Nontransformed (Ob1771) and polyoma virus-transformed (Ob17PY) mouse cells from the preadipocyte Ob17 clonal line have been compared in their ability to release prostaglandins in vitro as well as in vivo as assayed by in situ microdialysis. Prostaglandin FE2, prostaglandin-2 alpha and mainly prostacyclin are released in larger amounts (4- to 10-fold) by Ob17PY cells in vitro and Ob17PY-induced tumors in vivo as compared to Ob1771 preadipocytes in vitro and periepididymal adipose tissue in vivo. In contrast to Ob1771 preadipocytes, none of these prostanoids appear to be involved in the control of proliferation or differentiation of Ob17PY cells in serum-free culture medium. However, prostacyclin, the level of which is the most affected by transformation, might be considered as a valuable indicator of fibrosarcoma development.

Amino acid and glucose transport in sarcolemmal vesicles from chick embryo heart.

Purified plasma membranes from chick embryo heart are shown to retain several functional properties of intact cardiac cells. (1) Muscarinic cholinergic receptors copurify with previously used cell-surface markers, i.e., (K+-dependent) p-nitrophenylphosphatase and insulin receptors (Paris, S., Fosset, M., Samuel, D. and Ailhaud, G. (1977) J. Mol. Cell. Cardiol. 9, 161-174). (2) Neutral amino acids (L-alanine and alpha-aminoisobutyric acid) are actively transported into these osmotically active vesicles when an Na+ electrochemical gradient is imposed. The affinity and specificity for amino acids are similar to those described for intact cardiac cells. (3) D-Glucose is taken up more rapidly than L-glucose. The sterospecific transport system is saturable and Na+-independent. The Km value is close to that observed in intact cells with glucose analogues.

A continuous flow method for the study of lipoprotein lipase secretion in adipose cells.

The secretion of lipoprotein lipase has been examined in Ob17 adipose cells. No spontaneous secretion is detected. The activity of the heparin-releasable enzyme shows a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevents any significant determination of enzyme secretion in heparin-containing media. Thus, a perifusion system, with which the rate of enzyme inactivation is minimal and systematic, has been devised and used. The data show that the secretion of a pool of pre-existing lipoprotein lipase molecules is followed by the secretion of newly synthesized enzyme molecules. The results are discussed with respect to the significance of the determinations of the heparin-releasable enzyme in most studies as well as with respect to the intracellular localization of lipoprotein lipase in Ob17 cells.

Intracellular localization of lipoprotein lipase in adipose cells.

Subcellular localization of lipoprotein lipase has been examined in differentiated Ob17 adipose cells. No patent activity is detectable in carefully homogenized cells. All latent activity can be unmasked by disrupting membrane structures with neutral detergents. The sequestration of lipoprotein lipase in closed membrane structures is supported by experiments of immunotitration with anti-lipoprotein lipase antibodies and by experiments showing a full protection of the masked activity against proteolytic attack by trypsin. The intracellular distribution of lipoprotein lipase investigated by immunofluorescence staining and by isopycnic centrifugation indicates that a large proportion of the enzyme is located in the Golgi apparatus, in which the activation of the enzyme is likely to take place (C. Vannier et al. (1985) J. Biol. Chem. 260, 4424-4431). Altogether, the results are in favor of a localization of lipoprotein lipase in adipose cells as being typical of that of a secretory protein and underline the absence of lipoprotein lipase in the cell cytoplasm.

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.

Differentiation of ob 17 preadipocytes to adipocytes. Effects of prostaglandin F2alpha and relationship to prostaglandin synthesis.

The adipose conversion of ob 17 preadipose cells can be irreversibly blocked when prostaglandin F2alpha is included post-confluence for a minimum of 24 h in insulin-containing media. Prostaglandins E1 and E2 are inactive. The lack of adipose conversion is accompanied by the maintenance of a fusiform cell shape, by a slow increase in cell number and by a potent rise in de novo prostaglandin synthesis; it is paralleled by the absence of the characteristic phenotypes of adipose conversion. The multiple effects of prostaglandin F2alpha are dose-dependent, with half-maximal concentrations ranging from 10 to 40 nM. The absence of differentiation and the high rate of prostaglandin synthesis in the presence of prostaglandin F2alpha are likely a consequence of a sustained growth, as also observed with other growth-promoting agents (bovine retinal extract and cat serum). Indomethacin, while suppressing endogenous prostaglandin synthesis, is unable to reverse the long-term and multiple effects of prostaglandin F2alpha. Although adipose conversion normally follows a decrease in prostaglandin production (R. Négrel and G. Ailhaud, Biochem. Biophys. Res. Commun. (1981) 98, 768-777), these results indicate that both events can be dissociated.

Sugar transport in chick embryo cardiac cells in culture: analysis by countertransport, relationship to phosphorylation and effect of glucose starvation.

Embryonic chick heart cells in culture transport 2-deoxy-D-glucose and 3-O-methyl-D-glucose very rapidly. By direct measurements of uptake, it was not possible to estimate accurately transport rates, nor, with 2-deoxyglucose, to discriminate clearly between its transport and phosphorylation. In contrast, the technique of countertransport made it possible to determine precisely initial transport velocity and to make the following observations: (1) phosphorylation, and not transport, is rate-limiting in 2-deoxyglucose uptake; (2) hexose transport is stimulated 5-fold by removal of glucose from culture medium; and (3) this stimulation is followed by an increase in phosphorylation, but the effect is much less pronounced (2-fold stimulation only). In conclusion, the adaptative regulation of glucose transport described in many fibroblast cell lines exists also in cardiac cells.

Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells.

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells.

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.

Lipoprotein lipase and monoacylglycerol lipase activities during maturation of ob17 preadipocytes.

Lipoprotein lipase and monoacylglycerol lipase activities of developing ob17 preadipocytes were assayed through the selective inhibition of the former activity by 0.4% Triton X-100. After confluence both enzymes rose tao a peak activity around 13--15 days which was enhanced in insulin-treated cells. Inclusion of differentiation-blocking agents (bromodeoxyuridine, prostaglandin F2 alpha and bovine retinal extract) in the culture medium led in both enzymes to levels similar to those obtained in exponentially growing cells, in contrast to unspecific monoester hydrolase. Marked changes in the relative proportion of released to intracellular lipoprotein lipase activities were observed during adipose conversion. Our studies indicate that lipoprotein lipase and monoacylglycerol lipase are integral part of the program of differentiation of adipose cells.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. II. Effects of tunicamycin on activation and secretion of the enzyme.

The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells.

Autocrine control of adipose cell differentiation by prostacyclin and PGF2 alpha.

The mitogenic-adipogenic effect exerted by arachidonic acid, which leads to terminal differentiation of Ob1771 mouse preadipocytes, has been shown to be (i) blocked by cyclooxygenase inhibitors, (ii) mimicked by a stable analogue of prostacyclin (carbaprostacyclin) and (iii) potentiated by PGF2 alpha. Since these prostanoids are known to be synthesized and secreted by preadipocytes, we have proposed that both prostacyclin as the key mediator and PGF2 alpha as a modulator control the expression of terminal events of adipose conversion by means of an autocrine mechanism (Gaillard, D. et al. and Negrel, R. et al. Biochem. J. (1989) 257, 389-397 and 399-405). In order to test this hypothesis, the release of prostacyclin, characterized under the form of its stable degradation product 6-keto-PGF1 alpha, and that of PGF2 alpha have been studied in the culture medium of Ob1771 cells. A striking increase in the release of 6-keto-PGF1 alpha and to a minor degree of PGF2 alpha was observed when cells were exposed to arachidonic acid as shown by using [3H]arachidonic acid prelabelled cells or by radio-immunoassays. Since antagonists of PGF2 alpha and PGI2 receptors were not available, specific antibodies directed against PGF2 alpha and 6 beta-PGI1, another stable analogue of prostacyclin, were added as neutralizing agents in the culture medium. These antibodies were able to counteract the mitogenic-adipogenic effect of arachidonic acid. Prostacyclin and PGF2 alpha thus appear as autocrine mediators in the process of adipose conversion.

Differentiation of ob 17 preadipocytes to adipocytes. Triggering effects of clofenapate and indomethacin.

Conversion of ob 17 preadipocytes to mature adipose cells is accelerated by addition of clofenapate or of indomethacin, in either the absence or presence of insulin. General stimulation of triacylglycerol-pathway enzymes is observed, as well as dramatic increase in endogenous fatty-acid synthesis. This increase is a function of drug concentration and exposure time. In contrast to indomethacin, the continuous presence of clofenapate after the cells reached confluence was required to observe the effects on adipose conversion. Growth of ob 17 fibroblasts in the presence of 5-bromo-2'-deoxyuridine normally prevents their differentiation to adipose cells. Addition of either clofenapate or indomethacin to these cells at confluence overrides this block. The effects of hypolipidemic drugs such as clofenapate observed on a long-term basis in vitro are consistent with the results of studies on adipose tissue in vivo.

Apolipoprotein A-I-containing particles and reverse cholesterol transport in IDDM.

Lipoprotein particles containing apoA-I but not apoA-II are, among high-density lipoproteins, effective protectors against atherosclerosis that act by promoting the efflux of cellular cholesterol and the reverse cholesterol transport process. Because previous studies showed that in vitro nonenzymatic glycosylation of HDL impairs HDL receptor-mediated cholesterol efflux, we isolated Lp A-I from two poorly controlled insulin-dependent diabetic patients and compared the chemical composition and ability to promote cholesterol efflux with the same particles purified from two matched nondiabetic control subjects. No differences in lipid composition or in the ability to promote cholesterol efflux from cultured adipose cells were noted between the two types of Lp A-I preparations. However, when we separated Lp A-I from diabetic subjects by degree of glycosylation, the specifically glycosylated subfractions were about 50% less effective in producing cholesterol efflux than the nonglycosylated particles.

The human adipose tissue is a source of multipotent stem cells.

Multipotent stem cells constitute an unlimited source of differentiated cells that could be used in pharmacological studies and in medicine. Recently, several publications have reported that adipose tissue contains a population of cells able to differentiate into different cell types including adipocytes, osteoblasts, myoblasts, and chondroblasts. More recently, stem cells with a multi-lineage potential at the single cell level have been isolated from human adipose tissue. These cells, called human Multipotent Adipose-Derived Stem (hMADS) cells, have been established in culture and interestingly, maintain their characteristics with long-term passaging. The adipocyte differentiation of hMADS cells has been thoroughly studied and differentiated cells exhibit the unique feature of human adipocytes. Finally, potential applications of stem cells isolated from adipose tissue in medicine will be discussed.

Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation.

White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

Hormonal regulation of adipose differentiation.

Adipose differentiation is a multistep process with the following sequence: adipoblasts --> preadipocytes --> adipocytes. Adipogenic agents are only involved in the terminal differentiation of preadipocytes to adipocytes by means of circulating hormones (growth hormone, glucocorticoids, or triiodothyronine) and locally produced hormones (prostacyclin). Fatty acids also behave as hormones and act as transcriptional regulators of lipid-related genes. Once differentiated, adipocytes become secretory cells able to synthetize and release an impressive number of peptide and nonpeptide compounds, suggesting a potential link between excess of adipose tissue mass and various physiopathphysiologic consequences.

Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins beta and -delta in preadipocytes.

The essential role of CCAAT/enhancer binding proteins (C/EBPs) beta and delta for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP beta and C/EBP delta expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.