Adipogenesis In Preadipocytes Research Articles

Mature adipocytes inhibit in vitro differentiation of human preadipocytes via angiotensin type 1 receptors.

Recent studies suggest that angiotensin II (Ang II) plays a role in the adipogenesis of murine preadipocytes. Here, we examined the role of Ang II for the differentiation of primary cultured human preadipocytes. Preadipocytes were isolated from human adipose tissue and stimulated to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system (RAS) genes. The influence of the RAS on adipogenic differentiation was investigated by addition of either angiotensinogen (AGT), Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Expression of the RAS genes AGT, renin, angiotensin-converting enzyme, and Ang II type 1 receptor increased during adipogenesis. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture, and this effect was abolished by blockade of the Ang II type 1 receptor. This finding points to a functional role of the RAS in the differentiation of human adipose tissue. Because AGT secretion and Ang II generation are characteristic features of adipogenesis, we postulate a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes.

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways.

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.

Genes induced by growth hormone in a model of adipogenic differentiation

A substantial number of GH regulated genes have been reported in mature hepatocytes, but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a well-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2, Stat 3, thrombospondin-1, oncostatin M receptor β chain, a DEAD box RNA helicase, and muscleblind, a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine/threonine kinase. As each of the identified genes have important developmental roles, they may be important in initiating GH-induced adipogenesis.

Rapamycin inhibits human adipocyte differentiation in primary culture.

The immunosuppressant drug rapamycin, has been reported to inhibit 3T3-L1 adipocyte differentiation by interfering with critical postconfluent mitoses that are required early on for successful differentiation of this cell line (clonal expansion phase). In contrast to the murine 3T3-L1 preadipocyte cell line, human preadipocytes in primary culture do not undergo clonal expansion during differentiation. We investigated whether rapamycin could inhibit human adipocyte differentiation. The effect of rapamycin on the induction of differentiation of human preadipocytes in primary culture into adipocytes was measured using Oil Red O staining and glycerol phosphate dehydrogenase activity. We have observed that rapamycin severely curtails human adipocyte differentiation of both omental and abdominal subcutaneous preadipocytes (to 14% and 19% of standard differentiation, respectively). The rapamycin-mediated inhibition of human adipocyte differentiation could be reversed in the presence of excess amounts of FK-506, which displaces rapamycin from its intracellular receptor, FKPB12. Measurement of cytosolic protein and [3H]thymidine incorporation into DNA confirmed the absence of proliferation during differentiation of human preadipocytes in primary culture. Our data indicate that rapamycin exerts important negative regulatory effects on adipogenesis in human preadipocytes, through a mechanism that does not depend on interruption of clonal expansion.

Opposite Effects of Androgens and Estrogens on Adipogenesis in Rat Preadipocytes: Evidence for Sex and Site-Related Specificities and Possible Involvement of Insulin-Like Growth Factor 1 Receptor and Peroxisome Proliferator-Activated Receptor 2

To investigate the role of sex steroid hormones in adipose tissue development and distribution, we have studied the effect of various sex steroids (testosterone, dihydrotestosterone (DHT), and 17β-estradiol) in vitro, on the proliferation and differentiation processes in rat preadipocytes from deep (epididymal and parametrial) and superficial (femoral sc) fat deposits. All added steroids failed to affect the growth rate of preadipocytes from male rats when determined from day 1 to day 4 after plating, whether FCS was present or not in the culture medium. In contrast, in preadipocytes from female rats, we observed a positive effect (×2) of 17β-estradiol (0.01μ m) on the proliferative capacities of sc but not parametrial preadipocytes. When preadipocytes were exposed to testosterone or DHT (0.1 μm) during the differentiation process, the glycerol 3-phosphate dehydrogenase activity was significantly decreased in epididymal preadipocytes only. When preadipocytes from male rats were exposed to 17β-estradiol (0.01μ m), the differentiation capacities of preadipocytes were not modified. However, in parametrial preadipocytes from ovariectomized female rats, 17β-estradiol significantly increased (×1.34) the glycerol 3-phosphate dehydrogenase activity. In differentiated preadipocytes that had been exposed to sex steroids, expression of peroxisome proliferator-activated receptor γ2 was up-regulated by 17β-estradiol but not by androgens. As described in other cell types, sex steroids modulate insulin growth factor 1 receptor (IGF1R) expression in preadipocytes. Indeed, IGF1R levels were either enhanced by 17 β-estradiol (0.01 μm) in sc preadipocytes from female ovariectomized rats or decreased by DHT (0.01 μm) in epididymal preadipocytes. These effects were reversed by simultaneous exposure to androgen or estrogen receptor antagonists. In conclusion, this study demonstrates that, in rat preadipocytes kept in primary culture and chronically exposed to sex hormones, androgens elicit an antiadipogenic effect, whereas estrogens behave as proadipogenic hormones. Moreover, our results suggest that these opposite effects could be related to changes in IGF1R (androgens and estrogens) and peroxisome proliferator-activated receptor γ2 expression (estrogens).

Control of rat preadipocyte adipose conversion by ovarian status: regional specificity and possible involvement of the mitogen-activated protein kinase-dependent and c-fos signaling pathways.

As ovariectomy induces obesity in rats, we have investigated the influence of ovariectomy and hormone replacement on the proliferation and differentiation capacities of rat cultured preadipocytes removed from different fat depots (femoral sc, parametrial, and perirenal). Ovariectomy induced increased proliferation and differentiation as well as high mitogen-activated protein (MAP) kinase activity and c-fos protein induction in both confluent and differentiated preadipocytes from perirenal fat depots. In parametrial preadipocytes, ovariectomy also increased proliferation and c-fos protein induction, but failed to alter the capacities of these cells to differentiate. Treatment of ovariectomized rats with estradiol and progesterone reversed the promoting effect of ovariectomy on proliferation, differentiation, and c-fos induction in perirenal preadipocytes, but not the MAP kinase activation observed during the proliferative phase. This treatment also reversed the promoting effect of ovariectomy on proliferation and c-fos induction seen in confluent parametrial preadipocytes. In contrast, sc preadipocytes were totally insensitive to ovarian status in terms of proliferation and differentiation capacities, MAP kinase activity, and c-fos induction. This study demonstrates that adipogenesis is site-specifically controlled by the ovarian status in the rat. It also suggests that ovariectomy-induced obesity (mainly abdominal) could be related to changes in some of the signaling pathways controlling adipogenesis in intraabdominal preadipocytes.

Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis.

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.

Regulation of Growth Hormone (GH) Bioactivity by a Recombinant Human GH-Binding Protein*

The in vitro biological effects of serum GH-binding protein (GHBP) were measured in the mouse 3T3-F442A preadipocyte adipogenesis assay during GH stimulation. Coincubation of increasing concentrations of human (h) GH (0.14-4.5 nM) with 4.2 nM recombinant hGHBP-(1-247) in serum-free medium shifted the hGH dose-response curve to the right over the range 0.14-0.9 nM. When the hGH concentration was fixed at 0.45 nM, a dose-dependent inhibition of GH bioactivity was seen over the range of 0.1-11.3 nM GHBP, with an ED50 of 3 nM. The presence of serum had no effect on the inhibitory properties of GHBP. When 2% pooled human serum was added to incubation medium containing 0.45 nM hGH and GHBP (0.6 nM-5.7 nM), the effect of GHBP was again inhibitory, with an ED50 of 1.2 nM. Two percent serum alone was adipogenic, but at this low serum concentration it is likely that some factor other than GH is responsible. In a homologous receptor assay, the binding of [125I]hGH to IM-9 lymphocytes was inhibited in a dose-dependent manner by increasing concentrations of hGHBP in the physiological range, providing further support for the idea that GHBP can regulate the bioactivity of GH by blocking the binding of free GH to target tissues in vivo. Our results suggest that one function of GHBP is to dampen the biological effects of pulsatile GH secretion by reducing free GH during secretory pulses. This effect combined with an increased half-life of circulating GH would have the effect of flattening the hormone secretory profile at the target tissue level.