Thyroid hormone stimulates adipocyte differentiation of 3T3 cells

Triiodothyronine added at 0.1 nM to 3T3-F442A cells cultured in adipogenic medium having endogenous hormone concentrations similar to those of hypothyroid serum stimulated adipose conversion; activities of both lipogenic enzymes, glycerophosphate dehydrogenase and malic enzyme, increased with hormone treatment. The number of adipocytes was also augmented by L-T3 addition but the number of fat cell clusters remained the same as compared to non-treated cultures, suggesting that thyroid hormone increased the number of adipocytes probably through stimulating selective multiplication of precursor adipose cells. Hormone addition to cells cultured with non-adipogenic medium did not promote conversion showing that L-T3 is not an adipogenic factor by itself. Triiodothyronine added at concentrations similar to those found in hyperthyroidism, from 10 nM up to 10 µM, also increased the proportion of adipocytes without changing the number of fat cell clusters, but they decreased the activity of both lipogenic enzymes and lipid accumulation in mature adipocytes. It can be concluded that during 3T3-F442A differentiation into adipocytes L-T3 increases the number of differentiated adipocytes and, at low concentrations, also enhances lipogenic enzyme activities, whereas at the hyperthyroid hormone levels these enzyme activities are significantly reduced, remaining at levels similar to those of cells cultured with hypothyroid medium. This cloned cell line seems to be a useful model to study thyroid hormone action at both molecular and cellular level.     

Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IGF-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, [14C]glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and GPDH. The development of a serum-free, chemically defined medium for the differentiation of diploid adipose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.     

Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Decreased biosynthesis of actin and cellular fibronectin during adipose conversion of 3T3-F442A cells. Reorganization of the cytoarchitecture and extracellular matrix fibronectin

Differentiation of 3T3-F442A cells was accompanied by changes in cell morphology, decreased synthesis and assembly of actin and fibronectin. The network of microfilament stress fibers detected with NBD-phallacidin was altered during adipose conversion of 3T3-F442A cells. Parallel to this, the disappearance of fibrillar bundles of extracellular matrix fibronectin was observed by immunofluorescence staining. The pericellular fibronectin content, detected by immunoblotting, strongly diminished during the differentiation process. An altered rate of biosynthesis of both proteins was also measured by [35S]-methionine pulse-labeling and immunoprecipitation. A 4-5-fold decrease in cellular fibronectin synthesis was observed in adipocytes compared to control preadipocytes. Conversely, non-differentiating 3T3-C2 control cells did not reorganize either the cytoskeletal architecture or the extracellular matrix fibronectin in the resting state. These results suggest that the decreased rate of biosynthesis of cell-associated fibronectin is correlated with that of actin. Moreover, both events can essentially be ascribed to differentiation.     

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.     

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.     

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.     

Regulation of fibronectin, integrin and cytoskeleton expression in differentiating adipocytes: inhibition by extracellular matrix and polylysine

The differentiation of 3T3 preadipocytes into adipocytes is characterized by major changes in cell morphology from a fibroblastic to a rounded shape and by the induction of gene expression related to lipid metabolism. We have studied the synthesis and mRNA levels of proteins involved in the formation of cell-matrix contacts and in defining cell shape to determine the role and molecular basis of these morphological changes during adipose conversion. When confluent preadipocyte cultures were stimulated with adipogenic medium there was a gradual decrease in the expression of fibronectin, beta-integrin, actin and in the microfilament-associated proteins vinculin, alpha-actinin and tropomyosin. The changes in extracellular matrix and cytoskeletal mRNA levels were apparent before the accumulation of glycerophosphate dehydrogenase (GPD) mRNA and continued during the massive increase in GPD mRNA level. The culturing of preadipocytes on an extracellular matrix deposited on the dish by corneal endothelial cells, or on substrata coated with polylysine, prevented the morphological changes, the decrease in the level of assembled actin, the accumulation of lipid and the shifts in the expression of integrin, cytoskeletal proteins and GPD. In cells cultured on malleable hydrated collagen gels, adipocyte differentiation proceeded at normal rates. The results suggest that the regulated expression of proteins involved in the formation of the transmembrane linkage between the extracellular matrix and the microfilaments are programmed regulatory events that affect cell adhesion and thereby cell shape during adipocyte differentiation.     

Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells.

Besides nuclear retinoid receptors and cellular retinoid binding proteins also retinoic acid (RA)-synthesizing enzymes (using all-trans-retinal as substrate) and RA-catabolizing enzymes (producing hydroxylated products) may explain the specific effects of retinoids. In the past we have established an active role for 4-hydroxy-RA and 4-oxo-RA, which originally were considered to be inactive retinoids, but in fact are highly active modulators of positional specification in Xenopus development. Here we present evidence for a specific role of hydroxylated RA metabolites in the onset of neuronal differentiation. 4-Hydroxy- and 18-hydroxy-RA are products of the hydroxylation of RA by a novel cytochrome P450 (CYP)-type of enzyme, CYP26, expression of which is rapidly induced by RA. P19 embryonal carcinoma (EC) cell lines stably expressing hCYP26 undergo extensive and rapid neuronal differentiation in monolayer at already low concentrations of RA, while normally P19 cells under these conditions differentiate only in endoderm-like cells. Our results indicate that the effects on growth inhibition and RARbeta transactivation of P19 EC cells are mediated directly by RA, while the onset of neuronal differentiation and the subsequent expression of neuronal markers is mediated by hCYP26 via the conversion of RA to its hydroxylated products.     

Preadipocytes in the human subcutaneous adipose tissue display distinct features from the adult mesenchymal and hematopoietic stem cells

The stroma-vascular fraction (SVF) of human adipose tissue has recently been described to be composed of endothelial cells identified as CD34+/CD31+ cells, infiltrated/resident macrophages defined as CD14+/CD31+ cells, and a new cell population characterized as CD34+/CD31- cells. To elucidate the cell identity of the adipocyte precursor cells, fluorescent activating cell sorter (FACS) analyses were performed on crude SVF cultured under adipogenic conditions, i.e., serum-deprived medium containing insulin, cortisol, triiodothyronine, and supplemented with a PPARgamma agonist for the first 3 days. The progressive accumulation of lipid droplets was associated with a selective enrichment of the CD34+/CD31- cell population whereas control experiments performed in medium supplemented with 10% serum showed an overall downregulation of the three cell markers without adipogenesis. Among the different cell subsets, the CD34+/CD31- subset was the unique cell fraction able to answer to adipogenic culture conditions. Indeed, a time-dependent expression of adipocyte markers as well as acquisition of adipocyte-typical metabolic activities were observed. In parallel, the gene expression of lipogenic and lipolytic enzymes increased. The ability to differentiate into adipocytes was restricted to cells that did not express the mesenchymal stem cell marker CD105. Furthermore, the CD34+/CD31- cells did not respond to culture conditions used for hematopoietic colony assays. Taken together, the present study demonstrates that adipocyte progenitor cells, i.e., the preadipocytes, are included in the CD34+/CD31- cell fraction, which displays distinct features from the adult mesenchymal and hematopoietic stem cells.     

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

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

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.     

Dihydrocytochalasin B promotes adipose conversion of 3T3 cells

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

Inhibition of limb chondrogenesis by fibronectin

Abstract. This study compares the chondrogenic capacity of high density cultures prepared from either the develop-mentally younger, distal region or more advanced proximal region of stage 23/24 limb mesenchyme in high density cultures. Distal cultures undergo extensive chondrogenesis whether in F₁₂ medium supplemented with 10% fetal calf serum, 5% fetal calf serum, or fibronectin. On the other hand, proximal cultures fail to undergo chondrogenesis in medium containing 10% fetal calf serum or fibronectin, but do form cartilage in medium containing a decreased serum concentration or no serum. Furthermore, if the cells are cultured at low densities in native type I collagen gels, proximal cells have a reduced chondrogenic capacity in the presence of fibronectin, while chondrogenesis by distal cells is unaffected by the addition of fibronectin. The results demonstrate that proximal and distal cells respond differentially to serum and to fibronectin, and they suggest that the response of the cell to prevalent components of the extracellular matrix might change with development.     

Change of Morphology and Cytoskeletal Protein Gene Expression during Dibutyryl cAMP-induced Differentiation in C6 Glioma Cells

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of β-actin, γ-actin, and β-tubulin mRNAs in C6 cells, but not α-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except α-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.     

The tyrosine kinase receptor HER2 (erbB-2): from oncogenesis to adipogenesis

Recent experimental evidences begin to support the notion that the proto-oncogene HER2 (erbB-2) might unexpectedly function to modulate the adipogenic conversion of preadipocytes. Two opposing scenarios have been proposed, however, to explain the influence of HER2 on adipocyte differentiation. In one hand, down-modulation of HER2 expression and pharmacological reduction of HER2 activity have been related to enhanced adipocyte differentiation. On the contrary, an increased abundance in HER2 has been described in differentiated adipocytes compared with preadipocytes. Considering that expression and activity of the lipogenic enzyme Fatty Acid Synthase (FASN) become up-regulated during adipogenic conversion, we recently hypothesized that a "HER2 --> FASN axis" -a "lipogenic benefit" that has been shown to enhance cancer cell proliferation, survival, chemoresistance and metastasis in biologically aggressive subgroups of breast carcinomas-might also naturally work during the differentiation of preadipocytes. To definitely clarify if the discrepancy between the opposing theories for a role of HER2 during adipocyte differentiation related to the experimental approach utilized to compare the abundance of HER2 in undifferentiated and differentiated adipocytes (i.e., cell lysates containing equivalent protein content versus cell lysates generated from similar cell numbers), we here took advantage of a high content microscopy approach. Using an automated confocal imaging platform, we monitored the expression status of the adipogenic marker FASN and its timing relationship with HER2 not only in individual 3T3-L1 cells but further in whole cultures of 3T3-L1 preadipocytes undergoing adipogenic conversion. Our findings not only confirm a non-oncogenic role for HER2 in the process of adipose differentiation but further suggest that HER2 might represent a previously unrecognized target to manage obesity via the lipogenic enzyme FASN.     

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.     

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.