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.     

Suppression of the adipose conversion of 3T3 cells by acidified serum

The adipose conversion of cultured 3T3-F442A cells is strongly inhibited if the fetal bovine serum of the culture medium is briefly acidified before it is used. The inhibitory factor is a polypeptide with an apparent molecular weight of 24,000, and is inactivated by pronase or trypsin. Cells grown to confluence in the presence of this factor do not become spherical or accumulate triglyceride; they also do not increase the activity of their triglyceride-synthesizing enzymes. The factor suppresses adipose conversion even in the presence of untreated serum. Once adipose conversion has begun in the absence of the inhibitory factor, subsequent addition of the factor does not arrest the conversion.     

Adipose conversion of 3T3-L1 cells in a serum-free culture system depends on epidermal growth factor, insulin-like growth factor I, corticosterone, and cyclic AMP.

A culture system for 3T3-L1 preadipocytes based on a serum-free chemically defined medium containing fetuin, transferrin, and pantothenate is described. In this system, adipose conversion depends on the following conditions. 1) In the presence of high insulin concentrations (1 microM), addition of corticosterone together with 1-methyl-3-isobutylxanthine (MIX) for not more than the first 4 days after confluence to the culture medium induces maximal adipose conversion within 12-14 days. MIX may be replaced by forskolin or permeable analogues of cAMP, indicating that its effect is due to elevated cellular cAMP levels. 2) At low insulin concentrations (1 nM), adipose conversion is reduced. Growth hormone or insulin-like growth factor I together with epidermal growth factor have to be present as a medium supplement together with corticosterone and MIX to get maximal adipose conversion. 3) The induction of adipose conversion by corticosterone and MIX in the presence of either high insulin concentrations or insulin-like growth factor I together with epidermal growth factor is accompanied by post-confluent mitoses. Inhibitors of DNA replication markedly reduce adipose conversion. Fibroblast growth factor and platelet-derived growth factor, although acting as potent mitogens on 3T3-L1 cells, do not support adipose conversion induced by corticosterone and MIX.     

Studies of lipoprotein lipase during the adipose conversion of 3T3 cells.

Lipoprotein lipase activity is negligible in exponentially growing 3T3-L1 cells and 3T3-F442A cells, but develops in both lines when they reach a confluent state and undergo adipose conversion. 3T3-C2 cells, which undergo adipose conversion with extremely low frequency, do not develop the enzyme. The lipase activity of 3T3-L1 and 3T3-F442A is greatly enhanced by insulin and increases 80–180 fold during the adipose conversion. The lipase has the following characteristics in common with lipoprotein lipase from adipose and other tissues: it is dependent upon serum, is inhibited by 0.5–1.0 M sodium chloride, is recovered from acetone powders, has an alkaline pH optimum and is released from the cells by heparin. Like the lipoprotein lipase of tissue adipose cells, the enzyme of 3T3-L1 decays in the presence of cycloheximide with a half-time of about 25 min at 37°C.The ability of 3T3-F442A and 3T3-L1 to take up triglyceride from the medium depends almost completely upon lipoprotein lipase. They incorporate the fatty acids of a large fraction of a triglyceride emulsion added to the medium, and this utilization is stimulated by heparin. Very little of the glycerol portion of the triglyceride is incorporated. 3T3-C2, which lacks lipoprotein lipase, utilizes very little of either the fatty acid or the glycerol portion of triglyceride.The relevance of external lipid or lipoprotein to both the adipose conversion and the appearance of lipoprotein lipase was tested using confluent cultures in medium depleted of these components. In the presence of serum whose lipoproteins have been removed by flotation, lines 3T3-F442A and 3T3-L1 undergo adipose conversion as completely as in the presence of untreated serum, and lipoprotein lipase activity appears at essentially the same rate. In medium whose serum supplement has been extracted with acetone:ethanol, 3T3-F442A cells undergo adipose conversion to nearly the same extent as in untreated serum, and develop nearly the same increase in lipoprotein lipase activity.Unless even very low concentrations of lipids or lipoprotein are saturating it can be concluded that the adipose conversion does not depend upon external lipids or lipoproteins for its induction; rather the differentiation program is built into the cell type and comes into operation when growth is arrested even in their absence. The source of fatty acids utilized for triglyceride synthesis, however, may be affected by the amount of lipid provided to the cells.     

The induction of adipose conversion by bezafibrate in 3T3-L1 cells. Synergism with dibutyryl-cAMP

The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.     

Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells

When their growth is arrested in culture, susceptible 3T3 fibroblasts differentiate into adipose cells. Different clones form adipose cells with different frequency, depending upon the proportion of susceptible cells they contain. In cultures grown from small inocula, the fat cells appear in clusters formed by colonies of susceptible cells. Study of these clusters indicates the infrequent occurrence of cellular transitions from insusceptible to susceptible state.Beginning with a clone converting to adipose cells with a very low frequency, it has been possible, by serial selection, to generate subclones which convert with a high frequency. This evolution is due to spontaneous heritable changes affecting susceptibility to the adipose conversion. Presumably, they involve the control of triglyceride synthesis.Early stages of the adipose conversion may be recognized in stained cultures. When triglyceride first begins to accumulate, the highly extended and flattened processes of the cells are probably similar to those of nonfatty cells in the same cultures. As the adipose conversion proceeds, the processes thicken and retract; the cells eventually acquire the rounded shape of the more mature adipose cells.     

Interruption of the adipose conversion of 3T3 cells by biotin deficiency: differentiation without triglyceride accumulation

During the adipose conversion of 3T3 cells in medium supplemented with 10% serum, the accumulation of triglyceride depends upon a small molecular weight component in the serum. When this is removed by exhaustive dialysis of the serum, the cells undergo some changes that are part of the adipose conversion, but very little triglyceride accumulates. After the addition of either a serum dialysate or commercial biotin, cellular lipid begins to accumulate rapidly. The dialyzable factor in the serum has numerous chemical properties of biotin.When cells begin the adipose conversion in the absence of biotin, they are unable to increase their rate of acetate incorporation into triglyceride, but they do undergo the same change in shape to spherical, the same increase in activity of cytoplasmic (NAD-linked) glycerophosphate dehydrogenase (300 fold) and the same increase in lipoprotein lipase (>50 fold) as in the presence of biotin. On the other hand, in the biotin-deficient state the activity of glycerophosphate acyltransferase and of malic enzyme rises to only a small fraction of the control level. After the addition of biotin to the biotin-deficient cultures, the activity of both enzymes increases to the control level within 24–48 hr.These results are consistent with the concept that the glycerophosphate dehydrogenase is a primary enzyme in the adipose conversion: its response (measured as the amount of extractable activity) does not depend upon the rate of cellular fatty acid synthesis. Lipoprotein lipase is similarly independent. On the other hand, both malic enzyme and glycerophosphate acyltransferase may be considered as secondary enzymes in the sense that their response during the adipose conversion is linked to the supply of fatty acid.     

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.     

Commitment of adipocyte differentiation in 3T3 cells is inhibited by retinoic acid, and the expression of lipogenic enzymes is modulated through cytoskeleton stabilization

Retinoic acid (RA), at 1-10 microM, inhibited adipose conversion of 3T3-F442A cells as determined by the activities of lipogenic enzymes, glycerophosphate dehydrogenase (GPD) and malic enzyme. This inhibition was reversible by RA removal, but the increase in lipogenic enzyme activities was considerably delayed in a dose-dependent manner. The onset of the two lipogenic enzyme activities could be regulated somewhat independently, suggesting that expression of the two enzymes is subject to noncoordinated regulation. The RA-inhibited cells had a more flattened and elongated shape, suggesting cytoskeletal changes. Cytochalasin B (CB) did not prevent RA inhibition and did not promote adipose conversion in cultures supplemented with nonadipogenic medium. Reversion of inhibition was accelerated if cells were cultured for 3 days with adipogenic medium containing CB. The drug promoted an early increase in lipogenic enzyme activities. On the other hand, cells cultured on fibronectin-coated dishes, a condition that stabilizes actin cytoskeleton, do not undergo adipocyte differentiation. However, we show here that cells cultured on fibronectin and changed to nonadipogenic medium containing insulin underwent adipose conversion; in contrast, cells treated with RA and then supplemented with nonadipogenic medium containing insulin, but without the retinoid, did not undergo differentiation. We conclude that RA blocks adipose conversion probably before commitment to differentiation, and modulates lipogenic enzyme expression in a noncoordinated manner through changes in cytoskeletal elements, whereas fibronectin blocks phenotype expression in differentiating cells.     

Lipolysis and reesterification: effects of some inhibitors of adenosine 3',5'-cyclic monophosphate phosphodiesterase

Theophylline and three lipolytic agents, 2,5-bis(2-chloroethylsulfonyl)-pyrrole-3,4-dicarbonitrile (substituted pyrrole), 2,4-diamino-6-butoxy-s-triazine (substituted triazine), and 2,3-dihydro-5,6-dimethyl-3-oxo-4-pyridazinecarbonitrile (substituted pyridazine), stimulate basal lipolysis in adipose tissue in vitro. They also cause an increased release of free fatty acids, but not glycerol, from adipose tissue in which lipolysis is already maximally stimulated by epinephrine. The four compounds also inhibit cyclic AMP phosphodiesterase and the conversion of [1-(14)C]glucose to (14)CO(2). Evidence is presented that free fatty acids accumulate as the result of inhibited reesterification. The substituted pyridazine and triazine, but not the pyrrole, elevate plasma free fatty acids after oral or intraperitoneal administration in rats.     

Neutral and acid lipase of mouse 3T3 fibroblasts with increased adipose conversion.

In the resting state, 3T3-L1 fibroblasts become adipose converted and increase their fatty acid and triglyceride synthetase. We have found that they contain four times the neutral lipase activity and 1.5 times the acid lipase activity of logarithmically dividing cells. The activities of lysosomal acid beta-galactosidase and N-acetyl-beta-D-glucosaminidase were the same in the adipose converted and logarithmically dividing cells. The data suggest a possible relation between the increased neutral lipase activity in 3T3-L1 cells and their adipose conversion and demonstrates that the adipose converted 3T3-L1 fibroblasts, unlike true adipose cells, contain high levels of lysosomal acid hydrolases.     

Lithium ion reversibly inhibits inducer-stimulated adipose conversion of 3T3-L1 cells

Adipose conversion of 3T3-L1 cells by inducers (dexamethasone, 1-methyl-3-isobutylxanthine and insulin) was inhibited by LiCl at concentrations from 2 to 20 mM. The effect of LiCl was reversible and the inhibited cells were converted to adipocytes when stimulated after the removal of LiCl. Inhibition by LiCl of adipose conversion was accompanied with a blockage of the enhanced [3H]thymidine incorporation and cellular proliferation that occurred before the adipocyte phenotype was expressed. Of the cations tested, only Li+ had these effects.     

An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.

When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.     

Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.     

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.     

Dexamethasone regulation of terminal differentiation in 3T3-F442A preadipocyte cell line

The 3T3-F442A preadipocyte cell line was previously shown to possess specific glucocorticoid receptors whose number increased in the time course of differentiation. We have examined the effects of a three day dexamethasone treatment, added at confluence, on cells differentiated in the presence or absence of insulin. Triglyceride accumulation, polyamine content as well as glycerophosphate dehydrogenase and fatty acid synthetase activities were measured during the adipose conversion. We have also determined 2-deoxyglucose uptake in non-differentiated and differentiated cells. Dexamethasone was shown to decrease the adipose conversion by 3T3-F442A cells in the presence or absence of insulin. Intracellular spermidine content in differentiating cells was sensitive to dexamethasone and insulin in the same way as an enzymatic marker of terminal differentiation, glycerophosphate dehydrogenase. Dexamethasone decreases the 2 deoxyglucose uptake in non-differentiated and differentiated cells while insulin increases this uptake only in differentiated cells. This work shows that glucocorticoids inhibit adipocyte metabolism at distinct levels and suggests that these hormones might play an important role in the regulation of adipose tissue mass.Abbreviations DEX dexamethasone - FAS fatty acid synthetase - GPDH glycerophosphate dehydrogenase - MIX 1-methyl-3-isobutylxanthine