The agouti gene product stimulates pancreatic [beta]-cell Ca2+ signaling and insulin release.

Ubiquitous expression of the mouse agouti gene results in obesity and hyperinsulinemia. Human agouti is expressed in adipose tissue, and we found recombinant agouti protein to stimulate lipogenesis in adipocytes in a Ca(2+)-dependent fashion. However, adipocyte-specific agouti transgenic mice only became obese in the presence of hyperinsulinemia. Because intracellular Ca(2+) concentration ([Ca(2+)](i)) is a primary signal for insulin release, and we have shown agouti protein to increase [Ca(2+)](i) in several cell types, we examined the effects of agouti on [Ca(2+)](i) and insulin release. We demonstrated the expression of agouti in human pancreas and generated recombinant agouti to study its effects on Ca(2+) signaling and insulin release. Agouti (100 nM) stimulated Ca(2+) influx, [Ca(2+)](i) increase, and a marked stimulation of insulin release in two beta-cell lines (RIN-5F and HIT-T15; P < 0. 05). Agouti exerted comparable effects in isolated human pancreatic islets and beta-cells, with a 5-fold increase in Ca(2+) influx (P < 0.001) and a 2.2-fold increase in insulin release (P < 0.01). These data suggest a potential role for agouti in the development of hyperinsulinemia in humans.

Effects of a potent melanocortin agonist on the diabetic/obese phenotype in yellow mice.

OBJECTIVE: To test the hypothesis that a melanocortin agonist can reverse obesity and insulin resistance in mice overexpressing the agouti protein. EXPERIMENTAL MODEL: Mice overexpressing the agouti protein either by transgene introduction (beta-actin promotor) or by mutation (Ay). DESIGN: NDPMSH was tested for pharmacokinetic suitability. NDPMSH at various doses was administered subcutaneously twice a day for 2-3 weeks. MEASUREMENTS: Fur pigmentation, various fatness parameters (core temperature, fat pad weight and body weight), blood glucose and hormones, fatty acid synthase measurement. RESULTS: NDPMSH caused fur pigmentation and core temperature changes, but failed to affect any metabolic parameters in agouti-dependent manner. CONCLUSION: NDPMSH, as a representation melanocortin agonist, does not compete with agouti in reversing agouti-dependent metabolic effects. This suggests that 1) agouti works via a receptor other than a melanocortin receptor to mediate its metabolic effects, 2) agouti-dependent metabolic effects are mediated through melanocortin receptors but not via antagonism of these receptors, or 3) NDPMSH is pharmacodynamically an inappropriate molecule for these types of studies.

Angiotensinogen gene expression in adipose tissue: analysis of obese models and hormonal and nutritional control.

Synthesis of angiotensin II (ANG II) has recently been described in adipose cells and has been linked to regulation of adiposity. Angiotensinogen (AGT), the substrate from which ANG II is formed, was previously shown to be elevated in adipose tissue of obese (ob/ob and db/db) mice and regulated by nutritional manipulation. It is unknown, however, whether overexpression of adipose AGT can be extended to other models of obesity and whether hormonal and/or nutritional factors directly regulate AGT expression in adipocytes. We investigated these possibilities by analyzing AGT mRNA levels in adipose tissue of obese Zucker rats, viable yellow (Avy) mice, and humans and by treating 3T3-L1 adipocytes with insulin, glucose, and a beta-adrenergic agonist. We demonstrate that AGT mRNA is decreased by approximately 50 and 80%, respectively, in adipose tissue of obese vs. lean Zucker rats and Avy mice. We also report that AGT is expressed at variable levels in human adipose tissue. Finally, we show that AGT mRNA is upregulated by insulin and downregulated by beta-adrenergic stimulation in adipocytes.

Angiotensin II increases lipogenesis in 3T3-L1 and human adipose cells.

Angiotensin II (Ang II) is one of numerous hormones recently shown to be synthesized and secreted by adipose cells. Although the function of Ang II in adipose tissue is unknown, several studies indirectly suggest that it may be involved in control of adiposity. Little is known, however, about direct actions of Ang II in adipose cells. To further investigate this issue, we first characterized the type of Ang II receptors in 3T3-L1 adipocytes. We then tested the hypothesis that Ang II exerted direct actions on adipocyte metabolism using both 3T3-L1 and human adipocyte models. We report here that Ang II significantly increased triglyceride content and the activities of two key lipogenic enzymes (fatty acid synthase, FAS and glycerol-3-phosphate dehydrogenase, GPDH) in 3T3-L1 adipocytes, and that these effects were mediated through the type-2 Ang II receptor. We also report that Ang II exerted similar effects in human adipose cells maintained in primary culture. Finally, we demonstrate that Ang II increased the transcription rate of the FAS and ob genes in 3T3-L1 and human adipose cells. These results indicate that Ang II may be involved in control of adiposity through regulation of lipid synthesis and storage in adipocytes.

The effects of calcium channel blockade on agouti-induced obesity.

We have previously observed that obese viable yellow (Avy/a) mice exhibit increased intracellular Ca2+ ([Ca2+]i) and fatty acid synthase (FAS) gene expression; further, recombinant agouti protein increases in cultured adipocytes and these effects are inhibited by Ca2+ channel blockade. Accordingly, we determined the effect of Ca2+ channel blockade (nifedipine for 4 wk) on FAS and obesity in transgenic mice expressing the agouti gene in a ubiquitous manner. The transgenic mice initially were significantly heavier (30.5+/-0.6 vs. 27.3+/-0.3 g; P<0.001) and exhibited a 0.81 degrees C lower initial core temperature (P<0.0005), an approximately twofold increase in fat pad weights (P=0.002), a sevenfold increase in adipose FAS activity (P=0.009), and a twofold increase in plasma insulin level (P<0.05) compared to control mice. Nifedipine treatment resulted in an 18% decrease in fat pad weights (P<0.007) and a 74% decrease in adipose FAS activity (P=0.03), normalized circulating insulin levels and insulin sensitivity (P<0.05), and transiently elevated core temperature in the transgenic mice, but was without effect in the control mice. These data suggest that agouti regulates FAS, fat storage, and possibly thermogenesis, at least partially, via a [Ca2+]i-dependent mechanism, and that Ca2+ channel blockade may partially attenuate agouti-induced obesity.

Adipose tissue stearoyl-CoA desaturase mRNA is increased by obesity and decreased by polyunsaturated fatty acids.

Stearoyl-CoA desaturase (SCD) is a key regulatory enzyme in the synthesis of unsaturated fatty acids. Although regulation of hepatic SCD by obesity and polyunsaturated fatty acids (PUFA) has been well investigated, no studies have addressed whether similar regulation occurs in adipose tissue. We addressed these questions by feeding control (12% corn oil) and high-PUFA (48% corn oil) diets to lean and obese Zucker rats and analyzing SCD mRNA levels in adipose tissue and liver. We report that SCD mRNA content was dramatically elevated in adipose tissue of obese vs. lean rats on both diets and was significantly decreased by PUFA in both genotypes. Interestingly, we demonstrate that SCD expression was directly downregulated in a dose dependent manner by PUFA in 3T3-L1 adipocytes. We conclude that 1) obese Zucker rats overexpress the SCD gene in both liver and adipose tissue and 2) PUFA directly suppress SCD expression in adipocytes. Further studies will elucidate the mechanisms responsible for obesity- and PUFA-mediated regulation of SCD in adipose cells.

Insulin increases lipogenic enzyme activity in human adipocytes in primary culture.

Studies with human adipose tissue have demonstrated the presence of key enzymes of fat synthesis. However, long-term regulation of these enzymes has not been reported. To address this issue, we used human adipocytes in primary culture. Human adipose tissue was obtained from abdominal fat of patients undergoing abdominal surgery. Adipocytes were isolated by collagenase digestion and cultured in media supplemented with 1% fetal bovine serum. To evaluate metabolic activity of cultured cells, we assessed the following during the culture: DNA pattern, cell size, glucose consumption and activities for two lipogenic enzymes, fatty acid synthase (FAS) and glycerol-3-phosphate dehydrogenase (GPDH). Analysis of DNA pattern showed that human adipocytes cultured under the above condition did not undergo cell apoptosis. In addition, no significant change in the cell size occurred during 22 d of culture. Glucose consumption by cultured cells was also constant during the culture and was 60% greater in the presence of 10 nmol/L of insulin. Treatment of cultured human adipocytes with insulin for 3-22 d increased GPDH and FAS activity by 60% and 2.8-fold, respectively, compared to cells cultured without insulin. Furthermore, the increase in FAS activity due to insulin treatment was dose dependent and maximal at 10 nmol/L. Our studies show for the first time that human adipocytes can be maintained viable and metabolically active for 2-3 wk in culture. Interestingly, cultured cells remain responsive to insulin. Therefore, this system will allow further characterization of long-term regulation of lipogenesis in human adipocytes and will be useful for developing pharmacological treatments of obesity.

Upregulation of adipocyte metabolism by agouti protein: possible paracrine actions in yellow mouse obesity.

Mutations leading to ectopic expression of the murine agouti gene (a) result in progressive obesity. To further characterize this model, we analyzed adipose and hepatic mRNA levels for fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD), two key enzymes in de novo fatty acid synthesis and desaturation, respectively. FAS and SCD mRNA in both tissues of obese (Avy) mice were dramatically increased relative to lean (ala) controls. Excessive expression of these genes in this model could be due to direct effects of the agouti gene product; to test this possibility we treated 3T3-L1 adipocytes in vitro with recombinant agouti protein. Agouti treatment increased FAS and SCD mRNA levels by 1.5- and 4-fold, respectively. In addition, FAS activity and triglyceride content were 3-fold higher in agoutitreated 3T3-L1 cells relative to controls; these effects were attenuated by simultaneous treatment with a calcium channel blocker (nitrendipine). These data demonstrate that the agouti protein can directly increase lipogenesis in adipocytes and suggest that these effects are mediated through an intracellular calcium-dependent mechanism.

Localization of sequences for the basal and insulin-like growth factor-I inducible activity of the fatty acid synthase promoter in 3T3-L1 fibroblasts.

Fatty acid synthase (FAS) plays a central role in fatty acid synthesis and its expression is under nutritional and hormonal control. We have investigated insulin-like growth factor-I (IGF-I) regulation of FAS by transfecting into 3T3-L1 fibroblasts chimeric genes comprising the 5'-flanking region of the FAS gene linked to a luciferase (LUC) reporter gene. First, the basal promoter activity of the 5' serial deletions from nucleotides -318 to -19 of the FAS gene were compared. Deletions of the promoter sequences from -136 to -19 resulted in a step-wise decrease in the promoter activity, with the -67 LUC and -19 LUC plasmids retaining 40% and 16% of the luciferase activity of -136 LUC. Regulatory sequences important for the FAS basal promoter activity in 3T3-L1 fibroblasts are, therefore, located within the -136 to -19 region. Treatment with 10 mM IGF-I also increased luciferase activity 1.8 +/- 0.2-, 1.8 +/- 0.3- and 2.5 +/- 0.1-fold in 3T3-L1 fibroblasts transiently transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively. Deletion of sequences from -67 to -19 resulted in the loss of responsiveness to IGF-I. Physiological doses of insulin (10 nM), however, did not increase luciferase activity in 3T3-L1 fibroblasts transfected with any of the above plasmids. Only upon treatment with pharmacological doses of insulin (1 microM), probably through IGF-I receptor, did luciferase activity increase 4.3 +/- 0.4-, 3.2 +/- 0.4- and 3.5 +/- 0.5-fold when transfected with -136 LUC, -110 LUC and -67 LUC plasmids, respectively; there was no increase with -19 LUC. The half-maximal effect of IGF-I on FAS promoter activity was observed at 3 nM and a maximal effect was reached at 10 nM. These results indicate that the increased promoter activities observed are probably mediated through the IGF-I receptor. Furthermore, sequences responsible for IGF-I regulation of the FAS gene are located within the proximal promoter between nucleotides -67 and -19 of the FAS gene.

Identification of an insulin response element in the fatty acid synthase promoter.

We have previously reported that insulin increases fatty acid synthase (FAS) gene transcription, and that sequences responsible for positive regulation are located within the first 332 base pairs of the FAS promoter. To define minimal sequences required for insulin regulation within this region, chimeric constructs containing serial 5' deletions starting at -318 and extending through position +67 of the rat FAS gene ligated to the luciferase reporter gene were transfected into 3T3-L1 adipocytes. Insulin treatment at 10 nM increased luciferase activity 2-3-fold in 3T3-L1 adipocytes transfected with constructs containing progressive deletions from -318 to -67. This stimulation of the FAS promoter activity by insulin was dose-dependent. However, no effect of insulin was observed when fusion constructs containing FAS promoter sequences spanning from -25 or from -19 to +67 were transfected into adipocytes. These results suggest that the insulin response sequences of the FAS gene may be located in the region from -67 to -25. DNase I footprinting using liver nuclear extracts revealed a protected region spanning -71 and -50 in addition to a region near the putative TATA box. Gel mobility shift assays using the sequence from -71 to -50 as a probe revealed nuclear factor(s) from mouse liver and 3T3-L1 adipocytes that specifically complexed with this sequence. Mutational analysis of this region showed that sequences between -68 and -60 are essential for recognition and interaction with a trans-acting factor(s). Moreover, when three tandem repeats of the sequences spanning -68 to -52 were linked to the SV40 promoter and used for transfection, luciferase activity increased 3.6-fold in response to insulin treatment. Thus, we have identified novel cis-acting DNA sequences responsible for insulin regulation of the FAS gene, which interact with nuclear protein(s) from liver and adipocytes and which are found to share limited homology to insulin response sequences present in other genes.

Regulation of fatty acid synthase gene transcription. Sequences that confer a positive insulin effect and differentiation-dependent expression in 3T3-L1 preadipocytes are present in the 332 bp promoter.

We have previously reported induction of fatty acid synthase (FAS) gene expression by insulin and adipocyte differentiation in 3T3-L1 cells. In order to identify sequences responsible for insulin regulation of the FAS gene, chimaeric constructs containing serial deletions of the 5'-flanking region of the rat FAS gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were prepared and transfected into 3T3-L1 cells. Plasmids containing 2100 (-2100CAT), 1400 (-1400CAT), 1009 (-1009CAT) and 332 (-332CAT) bp of FAS 5' flanking sequences exhibited comparable basal CAT activities in 3T3-L1 preadipocytes. This activity was 3-fold higher when these constructs were transiently transfected into 3T3-L1 adipocytes. Stably transfected 3T3-L1 cells also exhibited a 3-fold increase in CAT activity upon adipocyte differentiation, indicating that sequences required for the differentiation-dependent increase in FAS expression are located within the 332 bp promoter. Treatment with 10 nM insulin increased CAT activity by 2.1 +/- 0.2-, 2.6 +/- 0.1-, 2.0 +/- 0.2- and 1.7 +/- 0.2-fold respectively in 3T3-L1 adipocytes transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids. CAT activity was increased by 3.0 +/- 0.3- and 3.5 +/- 0.6-fold respectively by insulin treatment in adipocytes stably transfected with -2100CAT and -1009CAT plasmids. When insulin-responsive H4IIE hepatoma cells were transiently transfected with -2100CAT, -1400CAT, -1009CAT and -332CAT plasmids and then treated with 10 nM insulin, CAT activity increased by 3.1-, 3.1 +/- 0.8-, 3.0 +/- 0.7- and 2.3 +/- 0.5-fold respectively in serum-free media, and by 2.6 +/- 0.4-, 3.3 +/- 0.9-, 3.1 +/- 0.4- and 2.9 +/- 0.6-fold respectively in the presence of 0.5% serum. These results indicate that sequences responsible for insulin regulation of FAS gene are also located within 332 bp of the transcription start site.

Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase.

We have previously isolated cDNA clones for several mRNAs that are dramatically increased in livers of fasted mice refed a high carbohydrate diet. We report here the sequence and regulation of one such mRNA; the 6.8-kilobase mRNA has an open reading frame of 2481 nucleotides, and the coded protein contains 827 amino acid residues (Mr of 90,000) with a 30% identity and an additional 42% similarity in an approximately 300-amino acid stretch to Escherichia coli glycerol-3-phosphate acyltransferase. The p90 mRNA is highly expressed in liver and in adipose tissue. When previously fasted mice were refed a high carbohydrate, fat-free diet, the liver mRNA level for p90 was increased about 20-fold at 8 h. Administration of dibutyryl cAMP at the time of refeeding prevented the increase in the p90 mRNA by 70%. In addition, there was no increase in the p90 mRNA level when previously starved streptozotocin-diabetic mice were refed. In diabetic animals, the p90 mRNA level increased by 2-fold 1 h after insulin injection and reached a maximum of 19-fold after 6 h. The increase in transcription rate of the p90 gene preceded that of steady state mRNA level caused by fasting/refeeding, and cAMP abolished the increase in transcription. Transcription of the p90 gene was not detectable in either fasted or refed streptozotocin-diabetic mice, but increased 4-fold 30 min after insulin administration and further increased up to 8-fold at 2 h. On-going protein synthesis was necessary for this increase.

Regulation of expression of the fatty acid synthase gene in 3T3-L1 cells by differentiation and triiodothyronine.

We have previously reported that fatty acid synthase mRNA levels increase 10-15-fold during the differentiation of 3T3-L1 cells to adipocytes, correlating well with the increase in the relative rate of synthesis for this enzyme. Here we show by transcription run-on assays that fatty acid synthase is highly transcribed in both preadipocytes and adipocytes. Furthermore, the transcription rate of the fatty acid synthase gene increased only 1.5-fold during the adipose conversion, whereas the apparent mRNA half-life increased from 2.5 h in preadipocytes to approximately 20 h in adipocytes. These results indicate that the increase in mRNA level during adipose conversion is not only due to the transcriptional activation of this gene but reflects the post-transcriptional stabilization of the message in adipocytes compared to preadipocytes. As thyroid hormone has been reported to increase lipogenic enzyme activities including fatty acid synthase in adipose tissue and in differentiating adipocytes in vitro, we used fully differentiated 3T3-L1 adipocytes to study T3 regulation of mammalian fatty acid synthase expression. We measured the effect of T3 on the relative rate of protein synthesis, mRNA content, and transcription rate of this gene. When mature adipocytes were treated with 10 nM T3, the relative rate of synthesis of fatty acid synthase increased 1.9-fold at 6 h, and it reached a maximum of 2.9-fold at 12 h. In addition, Northern blot analysis showed that T3 increased the steady-state mRNA level for fatty acid synthase by 2.4-fold at 12 h and 4.5-fold at 24 h. Furthermore, run-on transcription analysis with isolated nuclei from cells treated with T3 showed that the transcription rate of the fatty acid synthase gene increased 4.1-fold after 6 h of T3 treatment and remained at the stimulated level for 24 h. These results demonstrate that the increase in transcription of the fatty acid synthase gene preceded that of the steady-state mRNA level, indicating that T3 regulates expression of fatty acid synthase primarily by modulating the transcription rate of the gene. In conclusion, while the differentiation-dependent increase in fatty acid synthase is mediated by both transcriptional and posttranscriptional processes, T3 regulation is primarily at the transcriptional level.

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes.

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.

Analysis of the glucocorticoid receptor during differentiation of 3T3-F442A preadipocyte cell line in culture.

A pure glucocorticoid agonist RU 28362 and the potent antagonist RU 38486 were compared with dexamethasone for the evolution and the molecular nature of the GR during insulin-dependent conversion of 3T3-F442A preadipocytes into mature cells. In the whole cell assay system, the affinity for preadipocyte GR was observed in the order RU 38486 greater than RU 28362 greater than dexamethasone. The GR complex was most stable in presence of dexamethasone followed by the antagonist RU 38486 = the agonist RU 28362. Similar results were obtained in mature adipocytes but the binding of RU 38486 was more equivocal. An insulin-dependent differentiation process did not alter any of these parameters but increased the number of GR nearly fivefold over a 2-week period. Ion-exchange analysis of the cytosolic receptor revealed that the differentiation process was not accompanied by the appearance of any novel or new forms of GR, contrary to the situation in the liver, since both RU 38486 and dexamethasone were bound to identical molecular species of GR. These data provide a defined system for further analysis of cellular receptor as a function of steroid, tissue, and species, contrary to the classical dogma where GR is generally thought to be identical as a passive vehicle for the steroid in all circumstances, and affinity for steroid is generally equated with receptor stability.

Analysis of gene expression during adipogenesis in 3T3-F442A preadipocytes: insulin and dexamethasone control.

In the present study, we have investigated dexamethasone and insulin regulation of the expression of adipose-specific mRNA, namely, glycerophosphate dehydrogenase (G3PDH) and adipsin, at different stages of differentiation. During adipose conversion, insulin promotes an accumulation of G3PDH mRNA which is linked to cell differentiation; in fully differentiated cells, insulin is not required to maintain G3PDH gene expression. Differentiating cells in serum deprived medium already exhibit, at day 1, a maximal amount of mRNA encoding for adipsin, which is tenfold decreased by 10 nM of insulin; insulin also exerts a negative effect on the abundance of adipsin mRNA in mature cells. This result indicates that adipsin appears to be a very early marker of adipose conversion, the gene expression of which is down-regulated by the presence of insulin. Dexamethasone (DEX) decreases the G3PDH message at all stages of adipose conversion, while it promotes the accumulation of adipsin mRNA mainly in differentiating cells. In DEX-treated adipocytes, the transcription efficiency of the G3PDH gene is not altered, and reduction to 50% of the message is due essentially to an approximately twofold decrease in its half-life.

[Decrease of gene expression of glycerophosphate dehydrogenase by dexamethasone in differentiated 3T3-F442A cells: antagonism with insulin and antiglucocorticoid RU38486]. / Diminution de l'expression du gène de la glycérophosphate déshydrogénase par la dexaméthasone, dans les cellules 3T3-F442A différenciées: antagonisme avec l'insuline et l'antiglucocorticoïde RU38486.

Preadipocyte subclones derived from mouse 3T3 cells differentiate into adipocytes; this differentiation is characterized by an increased activity of numerous enzymes required for triglyceride synthesis and/or mobilization. Among these enzymes, the role of glycerophosphate dehydrogenase in the differentiation process has been previously reported. In the present work, we studied the hormonal regulation of glycerophosphate dehydrogenase gene expression (G3PDH) in differentiated 3T3-F442A adipocytes. Dexamethasone (DEX) elicited a 50% decrease in both mRNA content and specific activity of G3PDH. This effect was due to a posttranscriptional event since DEX shortened the half life of the mRNA, whereas it did not modify the transcription rate of this gene. The DEX effect is specific to G3PDH, since the expression of another adipose-specific gene, namely adipsin, is not modified by DEX treatment. Insulin counteracts the inhibitory effect of DEX, mainly by stabilizing the mRNA encoding for G3PDH. The antiglucocorticoid RU38486 is able to reverse DEX inhibition. Latter phenomenon suggests that DEX action on G3PDH gene expression could be mediated by glucocorticoid receptors.

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.

Glucocorticoid binding during the differentiation of 3T3-F442A fibroblasts into adipocytes. A possible regulatory effect of insulin.

Until recently, few studies had been carried out on receptors for glucocorticoids in adipocytes, although the role of these steroids is considerable. In the present studies, we chose the pre-adipocyte line 3T3-F442A, which constitutes an excellent model for investigating the differentiation and function of adipocytes. Using a whole cell assay system, we showed the existence of a homogenous class of sites with the characteristics of glucocorticoid receptors, that is, high-affinity binding which is reversible, specific and saturable. Whatever the state of cellular differentiation, the affinity of the receptor for dexamethasone did not vary, although we observed an increase in the number of sites during differentiation. When cells were differentiated in the presence of insulin, there was a further increase in the binding capacity; moreover, insulin deprivation of such adipocytes caused a decrease in the number of sites. Our results therefore suggest that factors other than the glucocorticoids themselves influence dexamethasone binding. It is suggested that insulin plays a role in the regulation of the number of glucocorticoid receptors.