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.     

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.     

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.     

Control of the adipogenic differentiation of 3T3-F442A cells by retinoic acid, dexamethasone, and insulin: a topographic analysis

Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.     

Staurosporine rapidly commits 3T3-F442A cells to the formation of adipocytes by activation of GSK-3beta and mobilization of calcium

Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3beta was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl(2), which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.     

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.     

11β-hydroxysteroid Dehydrogenase 1 in Adipocytes: Expression is Differentiation-dependent and Hormonally Regulated

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) catalyses the reversible metabolism of physiological glucocorticoids (cortisol, corticosterone) to inactive metabolites (cortisone, 11-dehydrocorticosterone), thus regulating glucocorticoid access to receptors. 11β-HSD-1 expression is regulated during development and by hormones in a tissue specific manner. The enzyme is highly expressed in liver, where it may influence glucocorticoid action on fuel metabolism, processes also important in adipose tissue. Here we show that 11β-HSD-1 is expressed in white adipose tissue, in both the adipocyte and stromal/vascular compartments, and in the adipocyte cell lines 3T3-F442A and 3T3-L1. In these cells, 11β-HSD-1 expression is induced upon differentiation into adipocytes and is characteristic of a ‘late differentiation’ gene, with maximal expression 6-8 days after confluence is reached. In intact 3T3-F442A adipocytes the enzyme direction is predominantly 11β-reduction, activating inert glucocorticoids. The expression of 11β-HSD-1 mRNA is altered in fully differentiated 3T3-F442A adipocytes treated with insulin, dexamethasone or a combination of the hormones, in an identical manner to glycerol-3-phosphate dehydrogenase (GPDH) mRNA (encoding a key enzyme in triglyceride synthesis and a well-characterised marker of adipocyte differentiation). The demonstration of 11β-HSD-1 expression in adipocytes and its predominant reductase activity in intact 3T3-F442A adipocytes suggests that 11β-HSD-1 may play an important role in potentiating glucocorticoid action in these cells. 3T3-F442A and 3T3-L1 represent useful model systems in which to examine the factors which regulate 11β-HSD-1 gene expression and the role of 11β-HSD-1 in modulating glucocorticoid action in adipose tissue.     

Growth hormone-induced alteration of morphology and tubulin expression in 3T3 preadipose cells

Effects of growth hormone on morphology and cytoskeletal protein expression were examined in 3T3-F442A preadipocytes in serum-free medium. Between 2 and 5 days of culture 2 nM methionyl human growth hormone converted 3T3-F442A cells from a flat fibroblastic morphology to a rounded form with numerous membrane convolutions. Growth hormone treated cultures manifested a 30-40% reduction in cell volume. Growth hormone induced changes in morphology and volume preceded and were independent of lipogenesis. In cells treated with growth hormone, expression of alpha and beta-tubulin as determined by Western blotting was found to increase approximately 50% within 72 h as compared to untreated cells. After 7 days, tubulin levels in growth hormone treated cells were approximately 40% of control levels. This indicated that morphological changes and alteration of tubulin expression were signatures of growth hormone action on 3T3-F442A cells.     

Transformation of Ob17 cells promotes proliferation and differentiation of Ob17 preadipocytes via distinct extracellular intermediates

Conditioned serum-free medium of Ob17 cells transformed by the middle-T-only gene of polyoma virus (Ob17MT cells) is able to support growth and adipose conversion of the parental Ob17 cells. Conditioned media from 3T3-F442A cells (untransformed preadipocyte clonal line) and MTT4 cells (middle-T-transformed non-preadipocyte clonal line) are inactive. The serum-free conditioned medium of Ob17MT cells is also active on growth and adipose conversion of 3T3-F442A cells. The morphological differentiation of Ob17 cells is accompanied by the expression of early (lipoprotein lipase, LPL) and late (glycerol-3-phosphate dehydrogenase, GPDH) biochemical markers of adipose conversion. Bio-Gel P-60 chromatography and SDS-PAGE have allowed characterization of a mitogenic fraction of apparent MW approximately equal to 28 Kd distinct from an adipogenic fraction of apparent MW less than 10 Kd. This adipogenic fraction is only required for the acquisition of the GPDH activity and is therefore active on terminal differentiation.     

Sensitivity of preadipose 3T3 cells to growth hormone

Cultured preadipose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3-F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.     

Differential regulation of two glucose transporters by chronic growth hormone treatment of cultured 3T3-F442A adipose cells

New methods for the analysis of glucose transporters were used to analyze the molecular mechanisms involved in the insulin-antagonistic effects of growth hormone (GH), which is known as a diabetogenic hormone. The ability of GH to alter the number and mRNA levels of two different glucose transporters in cultured 3T3-F442A adipocytes was investigated using specific antibodies and cDNA probes. At concentrations of GH as low as 0.5 and 5 ng/ml and at incubation times as short as 4 h, GH decreased rates of 2-deoxyglucose uptake in 3T3-F442A adipocytes. 3-O-Methyl-D-glucose uptake was inhibited to an extent similar to that of 2-deoxyglucose uptake (60-80%) after a 24-h incubation with GH (500 ng/ml), indicating that GH inhibits glucose metabolism specifically at the step of glucose transport. To determine whether reduced rates of glucose transport might result from reduced numbers of glucose transporters, whole cell lysates were prepared from GH-treated cells and subjected to immunoblotting using antibodies that identify Glut 1 (HepG2/rat brain) and Glut 4 (muscle/adipose) transporters. GH caused a time- and dose-dependent decrease in the number of Glut 1 transporters in the cell. Northern and slot-blot analyses showed a GH-induced dose-dependent decrease in levels of Glut 1 mRNA. In contrast, levels of Glut 4 transporter and mRNA were unchanged by GH. These data suggest that GH regulates Glut 1 and Glut 4 transporters differentially and that it exerts its inhibitory effect on glucose uptake at least in part by decreasing the synthesis of Glut 1 transporters. These studies provide the first evidence that GH regulates a key gene in metabolic regulation and can interfere with gene expression.     

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     

Cyclic AMP-mediated control of lipogenic enzyme synthesis during adipose differentiation of 3T3 cells

During the adipose differentiation of 3T3-F442A cells, there is an increase in the synthesis of numerous proteins, including the lipogenic enzymes glycerophosphate dehydrogenase, fatty acid synthetase and malic enzyme. Agents that increase cAMP content (Dibutyryl cAMP, theophylline, and isoproterenol) are known to induce lipolysis in fat cells; but the same agents are shown here to reduce the synthesis of the lipogenic enzymes during adipose differentiation. The extent of reduction depends on the agent used and differs for the three enzymes; fatty acid synthetase is most sensitive and its synthesis can be suppressed completely. In contrast to their effects on lipogenic enzyme synthesis, these agents do not affect morphological changes or the synthesis of several other proteins, of which some increase and others (such as actin) decrease during the differentiation. The effects of the agents on the synthesis of lipogenic enzymes are not dependent on lipolysis, since they take place to the same degree in cells not permitted to accumulate triglyceride. Translation in vitro of mRNA isolated from cells treated with the agents promoting cAMP accumulation indicates that the levels of functional mRNA for lipogenic enzymes are reduced. We conclude that, in addition to its activation of lipolysis, cAMP reduces specifically mRNA accumulation for lipogenic enzymes. These results also demonstrate the independent control of morphological change and enzyme synthesis during adipose differentiation.     

A central role for hepatocyte growth factor in adipose tissue angiogenesis

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor produced in human adipose tissue. In this study, we use 3T3-F442A preadipocytes to study the contribution of HGF to angiogenesis in an in vivo fat pad development model. As observed for human adipocytes, HGF is synthesized and secreted by 3T3-F442A preadipocytes and mature adipocytes. HGF knockdown with small-interfering RNA reduced HGF mRNA expression 82.3 +/- 4.2% and protein secretion 82.9 +/- 1.4% from 3T3-F442A preadipocytes. Silencing of HGF resulted in a 70.5 +/- 19.0% reduction in endothelial progenitor cell migration to 3T3-F442A-conditioned medium in vitro. 3T3-F442A preadipocytes injected under the skin of mice form a fat pad containing mature, lipid-filled adipocytes and a functional vasculature. At 72 h postinjection, expression of the endothelial cell genes TIE-1 and platelet endothelial cell adhesion molecule (PECAM)-1 was decreased 94.4 +/- 2.2 and 91.5 +/- 2.5%, respectively, in 3T3-F442A fat pads with HGF silencing. Knockdown of HGF had no effect on differentiation of 3T3-F442A preadipocytes to mature adipocytes in vitro or in vivo. In developing fat pads under the skin of HGF overexpressing transgenic mice, TIE-1 and PECAM-1 mRNA was increased 16.5- and 21.4-fold, respectively, at 72 h postinjection. The increase in gene expression correlated with immunohistochemical evidence of endothelial cell migration in the developing fat pad. These data suggest that HGF has a central role in regulating angiogenesis in adipose tissue.     

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.